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1
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Butt H, Sathish S, London E, Lee Johnson T, Essawi K, Leonard A, Tisdale JF, Demirci S. Genome editing strategies for targeted correction of β-globin mutation in sickle cell disease: From bench to bedside. Mol Ther 2025; 33:2154-2171. [PMID: 40165374 DOI: 10.1016/j.ymthe.2025.03.047] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2025] [Revised: 03/22/2025] [Accepted: 03/26/2025] [Indexed: 04/02/2025] Open
Abstract
Sickle cell disease (SCD) includes a range of genotypes that result in a clinical syndrome, where abnormal red blood cell (RBC) physiology leads to widespread complications affecting nearly every organ system. Treatment strategies for SCD can be broadly categorized into disease-modifying therapies and those aimed toward a cure. Although several disease-modifying drugs have been approved, they do not fully address the complexity and severity of SCD. Recent advances in allogeneic transplantation and autologous gene therapy show promising outcomes in terms of efficacy and safety. While these approaches have improved the lives of many patients, achieving a durable and comprehensive cure for all remains challenging. To address this, gene-editing technologies, including zinc-finger nucleases, TALENs, CRISPR-Cas, base editing, and prime editing, have been explored both ex vivo and in vivo for targeted correction of the β-globin gene (HBB) in SCD. However, direct correction of HBB and its translation from the laboratory to the clinic presents ongoing limitations, with challenges involved in achieving robust mutation-correction efficiency, off-target effects, and high costs of therapies. The optimal strategy for curing SCD remains uncertain, but several promising approaches are emerging. This review touches on past, present, and future developments in HBB correction.
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Affiliation(s)
- Henna Butt
- Cellular and Molecular Therapeutics Branch (CMTB), National Heart Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20814, USA
| | - Shruti Sathish
- Cellular and Molecular Therapeutics Branch (CMTB), National Heart Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20814, USA
| | - Evan London
- Cellular and Molecular Therapeutics Branch (CMTB), National Heart Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20814, USA
| | - Taylor Lee Johnson
- Cellular and Molecular Therapeutics Branch (CMTB), National Heart Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20814, USA
| | - Khaled Essawi
- Department of Medical Laboratory Technology, College of Applied Medical Sciences, Jazan University, Gizan 45142, Saudi Arabia
| | - Alexis Leonard
- Cellular and Molecular Therapeutics Branch (CMTB), National Heart Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20814, USA; Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - John F Tisdale
- Cellular and Molecular Therapeutics Branch (CMTB), National Heart Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20814, USA.
| | - Selami Demirci
- Cellular and Molecular Therapeutics Branch (CMTB), National Heart Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20814, USA.
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2
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Yang L, Wei W, Yuan X, Guo E, Peng P, Wang J, Sun W. Targeting DNA Damage Repair to Enhance Antitumor Immunity in Radiotherapy: Mechanisms and Opportunities. Int J Mol Sci 2025; 26:3743. [PMID: 40332379 PMCID: PMC12027993 DOI: 10.3390/ijms26083743] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2025] [Revised: 04/07/2025] [Accepted: 04/13/2025] [Indexed: 05/08/2025] Open
Abstract
Radiotherapy is a standard cancer treatment that involves the induction of DNA damage. DNA damage repair (DDR) pathways maintain genomic integrity and make tumors resistant to radiotherapy and certain chemotherapies. In turn, DDR dysfunction results in cumulative DNA damage, leading to increased sensitivity for antitumor treatment. Moreover, radiotherapy has been shown to trigger antitumor immunity. Currently, immunotherapy has become a new and widely used standard strategy for treating a broad spectrum of tumor types. Notably, recent studies have demonstrated that DDR pathways play important roles in driving the response to immunotherapy. Herein, we review and discuss how DDR affects antitumor immunity induced by radiotherapy. Furthermore, we summarize the development of strategies for combining DDR inhibitors with radiotherapy and/or immunotherapy to enhance their efficacy against cancers.
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Affiliation(s)
| | | | | | | | | | | | - Wei Sun
- Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China; (L.Y.); (W.W.); (X.Y.); (E.G.); (P.P.); (J.W.)
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3
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Liu Q, Li X, Xu H, Luo Y, Cheng L, Liang J, He Y, Liu H, Fang J, Huang J. Therapeutic gene correction of HBB frameshift CD41-42 (-TCTT) deletion in human hematopoietic stem cells. ADVANCED BIOTECHNOLOGY 2025; 3:2. [PMID: 39883359 PMCID: PMC11740860 DOI: 10.1007/s44307-024-00053-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/10/2024] [Revised: 10/28/2024] [Accepted: 11/25/2024] [Indexed: 01/31/2025]
Abstract
Β-thalassemia is one of the global health burdens. The CD41-42 (-TCTT) mutation at HBB is the most prevalent pathogenic mutation of β-thalassemia in both China and Southeast Asia. Previous studies focused on repairing the HBB CD41-42 (-TCTT) mutation in β-thalassemia patient-specific induced pluripotent stem cells, which were subsequently differentiated into hematopoietic stem and progenitor cells (HSPCs) for transplantation. In this study, we directly applied the CRISPR/Cas9-based gene editing therapy to correct the HBB CD41-42 (-TCTT) mutation in patient-derived HSPCs. The effective editing induced by Cas9:sgRNA ribonucleoprotein and single-stranded oligodeoxynucleotides (ssODNs) was confirmed in HUDEP-2 cell lines harboring the HBB CD41-42 (-TCTT) mutation. Further correction of heterozygote and homozygote HBB CD41-42 (-TCTT) mutations in patient-derived HSPCs resulted in a 13.4-40.8% increase in the proportion of HBB-expressing (HBB +) cells following erythroid differentiation in vitro. At 16 weeks post-xenotransplantation of the edited HSPCs into coisogenic immunodeficient mice, the reparation efficiency in engrafted bone marrow was 17.21% ± 3.66%. Multiparameter flow cytometric analysis of the engrafted bone marrow showed an increase in the percentage of HBB + cells without impairing the ability of engraftment, self-renewal, and multilineage hematopoietic repopulation of HSPCs. For the safety evaluation, 103 potential off-target sites were predicted by SITE-seq and CRISPOR, with one site displaying significant off-target editing. Since this off-target site is located in the intergenic region, it is presumed to pose minimal risk. Taken together, our study provides critical preclinical data supporting the safety and efficacy of the gene therapy approach for HBB CD41-42 (-TCTT) mutation.
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Affiliation(s)
- Qianyi Liu
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Xinyu Li
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, No.107, West Yan Jiang Road, Guangzhou, 510120, Guangdong, China
| | - Hui Xu
- Reforgene Medicine, Guangzhou, 510535, Guangdong, China
| | - Ying Luo
- Reforgene Medicine, Guangzhou, 510535, Guangdong, China
| | - Lin Cheng
- Reforgene Medicine, Guangzhou, 510535, Guangdong, China
| | - Junbin Liang
- Reforgene Medicine, Guangzhou, 510535, Guangdong, China
| | - Yuelin He
- Dongguan Taixin Hospital, Dongguan, 523170, Guangdong, China
| | - Haiying Liu
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Jianpei Fang
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, No.107, West Yan Jiang Road, Guangzhou, 510120, Guangdong, China.
| | - Junjiu Huang
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China.
- Key Laboratory of Reproductive Medicine of Guangdong Province, the, First Affiliated Hospital and School of Life Sciences , Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China.
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4
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Joudeh LA, Schuck PL, Van NM, DiCintio AJ, Stewart JA, Waldman AS. Progerin can induce DNA damage in the absence of global changes in replication or cell proliferation. PLoS One 2024; 19:e0315084. [PMID: 39636792 PMCID: PMC11620420 DOI: 10.1371/journal.pone.0315084] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2024] [Accepted: 11/15/2024] [Indexed: 12/07/2024] Open
Abstract
Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, and individuals with HGPS seldom live beyond their mid-teens. The syndrome is commonly caused by a point mutation in the LMNA gene which codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The mutation causing HGPS leads to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs) and alterations in the nature of DSB repair. The source of DSBs in HGPS is often attributed to stalling and subsequent collapse of replication forks in conjunction with faulty recruitment of repair factors to damage sites. In this work, we used a model system involving immortalized human cell lines to investigate progerin-induced genomic damage. Using an immunofluorescence approach to visualize phosphorylated histone H2AX foci which mark sites of genomic damage, we report that cells engineered to express progerin displayed a significant elevation of endogenous damage in the absence of any change in the cell cycle profile or doubling time of cells. Genomic damage was enhanced and persistent in progerin-expressing cells treated with hydroxyurea. Overexpression of wild-type lamin A did not elicit the outcomes associated with progerin expression. Our results show that DNA damage caused by progerin can occur independently from global changes in replication or cell proliferation.
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Affiliation(s)
- Liza A. Joudeh
- Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America
| | - P. Logan Schuck
- Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America
| | - Nina M. Van
- Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America
| | - Alannah J. DiCintio
- Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America
| | - Jason A. Stewart
- Department of Biology, Western Kentucky University, Bowling Green, Kentucky, United States of America
| | - Alan S. Waldman
- Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America
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5
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Cullati SN, Akizuki K, Shan Y, Zhang E, Ren L, Guillen RX, Turner LA, Chen JS, Navarrete-Perea J, Elmore ZC, Gygi SP, Gould KL. The DNA Damage Repair Function of Fission Yeast CK1 Involves Targeting Arp8, a Subunit of the INO80 Chromatin Remodeling Complex. Mol Cell Biol 2024; 44:562-576. [PMID: 39387272 PMCID: PMC11583621 DOI: 10.1080/10985549.2024.2408016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2024] [Revised: 09/10/2024] [Accepted: 09/18/2024] [Indexed: 10/15/2024] Open
Abstract
The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells increased double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and nonhomologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. To understand how Hhp1 and Hhp2 promote DNA damage repair, we identified new substrates of these enzymes using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 important for DNA repair. Our data suggest that Hhp1 and Hhp2 facilitate DNA repair by phosphorylating multiple substrates, including Arp8.
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Affiliation(s)
- Sierra N. Cullati
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Kazutoshi Akizuki
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Yufan Shan
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Eric Zhang
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Liping Ren
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Rodrigo X. Guillen
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Lesley A. Turner
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Jun-Song Chen
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | | | - Zachary C. Elmore
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Steven P. Gygi
- Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA
| | - Kathleen L. Gould
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
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6
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Yang K, Zhu L, Liu C, Zhou D, Zhu Z, Xu N, Li W. Current status and prospect of the DNA double-strand break repair pathway in colorectal cancer development and treatment. Biochim Biophys Acta Mol Basis Dis 2024; 1870:167438. [PMID: 39059591 DOI: 10.1016/j.bbadis.2024.167438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2023] [Revised: 07/18/2024] [Accepted: 07/18/2024] [Indexed: 07/28/2024]
Abstract
Colorectal cancer (CRC) is one of the most common malignancies worldwide. Double-strand break (DSB) is the most severe type of DNA damage. However, few reviews have thoroughly examined the involvement of DSB in CRC. Latest researches demonstrated that DSB repair plays an important role in CRC. For example, DSB-related genes such as BRCA1, Ku-70 and DNA polymerase theta (POLQ) are associated with the occurrence of CRC, and POLQ even showed to affect the prognosis and resistance for radiotherapy in CRC. This review comprehensively summarizes the DSB role in CRC, explores the mechanisms and discusses the association with CRC treatment. Four pathways for DSB have been demonstrated. 1. Nonhomologous end joining (NHEJ) is the major pathway. Its core genes including Ku70 and Ku80 bind to broken ends and recruit repair factors to form a complex that mediates the connection of DNA breaks. 2. Homologous recombination (HR) is another important pathway. Its key genes including BRCA1 and BRCA2 are involved in finding, pairing, and joining broken ends, and ensure the restoration of breaks in a normal double-stranded DNA structure. 3. Single-strand annealing (SSA) pathway, and 4. POLθ-mediated end-joining (alt-EJ) is a backup pathway. This paper elucidates roles of the DSB repair pathways in CRC, which could contribute to the development of potential new treatment approaches and provide new opportunities for CRC treatment and more individualized treatment options based on therapeutic strategies targeting these DNA repair pathways.
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Affiliation(s)
- Kexin Yang
- Department of Colorectal Surgery, the Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital, Kunming 650106, China; Kunming Medical University, Kunming 650500, China
| | - Lihua Zhu
- Department of Surgical Oncology, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China; Kunming Medical University, Kunming 650500, China
| | - Chang Liu
- Department of Surgical Oncology, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China
| | - Dayang Zhou
- Department of Surgical Oncology, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China
| | - Zhu Zhu
- Department of Surgical Oncology, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China
| | - Ning Xu
- Department of Colorectal Surgery, the Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital, Kunming 650106, China; Department of Surgical Oncology, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China; Kunming Medical University, Kunming 650500, China.
| | - Wenliang Li
- Department of Colorectal Surgery, the Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital, Kunming 650106, China; Kunming Medical University, Kunming 650500, China.
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7
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Fritsche S, Reinfurt A, Fronek F, Steiger MG. NHEJ and HDR can occur simultaneously during gene integration into the genome of Aspergillus niger. Fungal Biol Biotechnol 2024; 11:10. [PMID: 39103967 DOI: 10.1186/s40694-024-00180-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Accepted: 07/07/2024] [Indexed: 08/07/2024] Open
Abstract
Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are two mechanisms in filamentous fungi to repair DNA damages. NHEJ is the dominant response pathway to rapidly join DNA double-strand breaks, but often leads to insertions or deletions. On the other hand, HDR is more precise and utilizes a homologous DNA template to restore the damaged sequence. Both types are exploited in genetic engineering approaches ranging from knock-out mutations to precise sequence modifications.In this study, we evaluated the efficiency of an HDR based gene integration system designed for the pyrG locus of Aspergillus niger. While gene integration was achieved at a rate of 91.4%, we also discovered a mixed-type repair (MTR) mechanism with simultaneous repair of a Cas9-mediated double-strand break by both NHEJ and HDR. In 20.3% of the analyzed transformants the donor DNA was integrated by NHEJ at the 3' end and by HDR at the 5' end of the double-strand break. Furthermore, sequencing of the locus revealed different DNA repair mechanisms at the site of the NHEJ event.Together, the results support the applicability of the genome integration system and a novel DNA repair type with implication on the diversity of genetic modifications in filamentous fungi.
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Affiliation(s)
- Susanne Fritsche
- Austrian Centre of Industrial Biotechnology, Muthgasse 18, Vienna, Austria
- Institute of Chemical, Environmental and Bioscience Engineering, Research Group Biochemistry, Technische Universität Wien, Gumpendorferstrasse 1A, Vienna, 1060, Austria
| | - Aline Reinfurt
- Austrian Centre of Industrial Biotechnology, Muthgasse 18, Vienna, Austria
- Institute of Chemical, Environmental and Bioscience Engineering, Research Group Biochemistry, Technische Universität Wien, Gumpendorferstrasse 1A, Vienna, 1060, Austria
| | - Felix Fronek
- Austrian Centre of Industrial Biotechnology, Muthgasse 18, Vienna, Austria
- Institute of Chemical, Environmental and Bioscience Engineering, Research Group Biochemistry, Technische Universität Wien, Gumpendorferstrasse 1A, Vienna, 1060, Austria
| | - Matthias G Steiger
- Austrian Centre of Industrial Biotechnology, Muthgasse 18, Vienna, Austria.
- Institute of Chemical, Environmental and Bioscience Engineering, Research Group Biochemistry, Technische Universität Wien, Gumpendorferstrasse 1A, Vienna, 1060, Austria.
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8
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Joudeh LA, Logan Schuck P, Van NM, DiCintio AJ, Stewart JA, Waldman AS. Progerin Can Induce DNA Damage in the Absence of Global Changes in Replication or Cell Proliferation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.07.02.601729. [PMID: 39005395 PMCID: PMC11244969 DOI: 10.1101/2024.07.02.601729] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/16/2024]
Abstract
Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, and individuals with HGPS seldom live beyond their mid-teens. The syndrome is commonly caused by a point mutation in the LMNA gene which codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The mutation causing HGPS leads to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs) and alterations in the nature of DSB repair. The source of DSBs in HGPS is often attributed to stalling and subsequent collapse of replication forks in conjunction with faulty recruitment of repair factors to damage sites. In this work, we used a model system involving immortalized human cell lines to investigate progerin-induced genomic damage. Using an immunofluorescence approach to visualize phosphorylated histone H2AX foci which mark sites of genomic damage, we report that cells engineered to express progerin displayed a significant elevation of endogenous damage in the absence of any change in the cell cycle profile or doubling time of cells. Genomic damage was enhanced and persistent in progerin-expressing cells treated with hydroxyurea. Overexpression of wild-type lamin A did not elicit the outcomes associated with progerin expression. Our results show that DNA damage caused by progerin can occur independently from global changes in replication or cell proliferation.
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Affiliation(s)
- Liza A. Joudeh
- Department of Biological Sciences, University of South Carolina, Columbia, SC 20208
| | - P. Logan Schuck
- Department of Biological Sciences, University of South Carolina, Columbia, SC 20208
| | - Nina M. Van
- Department of Biological Sciences, University of South Carolina, Columbia, SC 20208
| | - Alannah J. DiCintio
- Department of Biological Sciences, University of South Carolina, Columbia, SC 20208
| | - Jason A. Stewart
- Department of Biological Sciences, University of South Carolina, Columbia, SC 20208
- Department of Biology, Western Kentucky University, Bowling Green, KY 42101
| | - Alan S. Waldman
- Department of Biological Sciences, University of South Carolina, Columbia, SC 20208
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9
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Coll RP, Bright SJ, Martinus DKJ, Georgiou DK, Sawakuchi GO, Manning HC. Alpha Particle-Emitting Radiopharmaceuticals as Cancer Therapy: Biological Basis, Current Status, and Future Outlook for Therapeutics Discovery. Mol Imaging Biol 2023; 25:991-1019. [PMID: 37845582 PMCID: PMC12054971 DOI: 10.1007/s11307-023-01857-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2023] [Revised: 09/03/2023] [Accepted: 09/05/2023] [Indexed: 10/18/2023]
Abstract
Critical advances in radionuclide therapy have led to encouraging new options for cancer treatment through the pairing of clinically useful radiation-emitting radionuclides and innovative pharmaceutical discovery. Of the various subatomic particles used in therapeutic radiopharmaceuticals, alpha (α) particles show great promise owing to their relatively large size, delivered energy, finite pathlength, and resulting ionization density. This review discusses the therapeutic benefits of α-emitting radiopharmaceuticals and their pairing with appropriate diagnostics, resulting in innovative "theranostic" platforms. Herein, the current landscape of α particle-emitting radionuclides is described with an emphasis on their use in theranostic development for cancer treatment. Commonly studied radionuclides are introduced and recent efforts towards their production for research and clinical use are described. The growing popularity of these radionuclides is explained through summarizing the biological effects of α radiation on cancer cells, which include DNA damage, activation of discrete cell death programs, and downstream immune responses. Examples of efficient α-theranostic design are described with an emphasis on strategies that lead to cellular internalization and the targeting of proteins involved in therapeutic resistance. Historical barriers to the clinical deployment of α-theranostic radiopharmaceuticals are also discussed. Recent progress towards addressing these challenges is presented along with examples of incorporating α-particle therapy in pharmaceutical platforms that can be easily converted into diagnostic counterparts.
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Affiliation(s)
- Ryan P Coll
- Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, 1881 East Rd, Houston, TX, 77054, USA
| | - Scott J Bright
- Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, 6565 MD Anderson Blvd, Houston, TX, 77030, USA
| | - David K J Martinus
- Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, 6565 MD Anderson Blvd, Houston, TX, 77030, USA
| | - Dimitra K Georgiou
- Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, 1881 East Rd, Houston, TX, 77054, USA
| | - Gabriel O Sawakuchi
- Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, 6565 MD Anderson Blvd, Houston, TX, 77030, USA
| | - H Charles Manning
- Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, 1881 East Rd, Houston, TX, 77054, USA.
- Cyclotron Radiochemistry Facility, The University of Texas MD Anderson Cancer Center, 1881 East Rd, Houston, TX, 77054, USA.
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10
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Deshpande RA, Marin-Gonzalez A, Barnes HK, Woolley PR, Ha T, Paull TT. Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice. Nat Commun 2023; 14:5759. [PMID: 37717054 PMCID: PMC10505227 DOI: 10.1038/s41467-023-41544-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2022] [Accepted: 09/07/2023] [Indexed: 09/18/2023] Open
Abstract
The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5' strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site-a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate sites of MRN-dependent processing by identifying sites of CtIP association and by sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated most efficiently when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that both complexes bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.
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Affiliation(s)
| | - Alberto Marin-Gonzalez
- Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA
- Howard Hughes Medical Institute, Baltimore, MD, 21205, USA
| | - Hannah K Barnes
- The Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, 78712, USA
| | - Phillip R Woolley
- The Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, 78712, USA
| | - Taekjip Ha
- Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA
- Howard Hughes Medical Institute, Baltimore, MD, 21205, USA
| | - Tanya T Paull
- The Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, 78712, USA.
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11
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Ghosh I, Kwon Y, Shabestari AB, Chikhale R, Chen J, Wiese C, Sung P, De Benedetti A. TLK1-mediated RAD54 phosphorylation spatio-temporally regulates Homologous Recombination Repair. Nucleic Acids Res 2023; 51:8643-8662. [PMID: 37439356 PMCID: PMC10484734 DOI: 10.1093/nar/gkad589] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2022] [Revised: 05/17/2023] [Accepted: 06/28/2023] [Indexed: 07/14/2023] Open
Abstract
Environmental agents like ionizing radiation (IR) and chemotherapeutic drugs can cause severe damage to the DNA, often in the form of double-strand breaks (DSBs). Remaining unrepaired, DSBs can lead to chromosomal rearrangements, and cell death. One major error-free pathway to repair DSBs is homologous recombination repair (HRR). Tousled-like kinase 1 (TLK1), a Ser/Thr kinase that regulates the DNA damage checkpoint, has been found to interact with RAD54, a central DNA translocase in HRR. To determine how TLK1 regulates RAD54, we inhibited or depleted TLK1 and tested how this impacts HRR in human cells using a ISce-I-GR-DsRed fused reporter endonuclease. Our results show that TLK1 phosphorylates RAD54 at three threonines (T41, T59 and T700), two of which are located within its N-terminal domain (NTD) and one is located within its C-terminal domain (CTD). Phosphorylation at both T41 and T59 supports HRR and protects cells from DNA DSB damage. In contrast, phosphorylation of T700 leads to impaired HRR and engenders no protection to cells from cytotoxicity and rather results in repair delay. Further, our work enlightens the effect of RAD54-T700 (RAD54-CTD) phosphorylation by TLK1 in mammalian system and reveals a new site of interaction with RAD51.
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Affiliation(s)
- Ishita Ghosh
- Department of Biochemistry and Molecular Biology, Louisiana Health Science Center-Shreveport, Shreveport, Louisiana 71130, US2. Texas 78229, USA
| | - Youngho Kwon
- Department of Biochemistry & Structural Biology, Greehey Children's Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78229, USA
| | - Aida Badamchi Shabestari
- Department of Biochemistry & Structural Biology, Greehey Children's Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78229, USA
| | - Rupesh Chikhale
- Division of Pharmacy & Optometry, University of Manchester, Manchester, UK
| | - Jing Chen
- Department of Molecular and Cellular Biochemistry and Proteomics Core, Center for Structural Biology, University of Kentucky, Lexington, KY, USA
| | - Claudia Wiese
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
| | - Patrick Sung
- Department of Biochemistry & Structural Biology, Greehey Children's Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78229, USA
| | - Arrigo De Benedetti
- Department of Biochemistry and Molecular Biology, Louisiana Health Science Center-Shreveport, Shreveport, Louisiana 71130, US2. Texas 78229, USA
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12
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Mikkelsen NS, Hernandez SS, Jensen TI, Schneller JL, Bak RO. Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene. Mol Ther Methods Clin Dev 2023; 29:1-16. [PMID: 36922985 PMCID: PMC10009645 DOI: 10.1016/j.omtm.2023.02.010] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2022] [Accepted: 02/13/2023] [Indexed: 02/18/2023]
Abstract
CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.
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Affiliation(s)
| | | | - Trine I Jensen
- Department of Biomedicine, Aarhus University, Aarhus C, Denmark
| | - Jessica L Schneller
- Department of Biomedicine, Aarhus University, Aarhus C, Denmark.,RNA and Gene Therapies, Novo Nordisk A/S, Maaloev, Denmark
| | - Rasmus O Bak
- Department of Biomedicine, Aarhus University, Aarhus C, Denmark.,Aarhus Institute of Advanced Studies, Aarhus University, Aarhus C, Denmark
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13
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Joudeh LA, DiCintio AJ, Ries MR, Gasperson AS, Griffin KE, Robbins VP, Bonner M, Nolan S, Black E, Waldman AS. Corruption of DNA end-joining in mammalian chromosomes by progerin expression. DNA Repair (Amst) 2023; 126:103491. [PMID: 37018982 PMCID: PMC10133198 DOI: 10.1016/j.dnarep.2023.103491] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Revised: 03/23/2023] [Accepted: 03/29/2023] [Indexed: 04/03/2023]
Abstract
Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging and a life expectancy of about 14 years. HGPS is commonly caused by a point mutation in the LMNA gene which codes for lamin A, an essential component of the nuclear lamina. The HGPS mutation alters splicing of the LMNA transcript, leading to a truncated, farnesylated form of lamin A termed "progerin." Progerin is also produced in small amounts in healthy individuals by alternative splicing of RNA and has been implicated in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs), suggesting alteration of DNA repair. DSB repair normally occurs by either homologous recombination (HR), an accurate, templated form of repair, or by nonhomologous end-joining (NHEJ), a non-templated rejoining of DNA ends that can be error-prone; however a good portion of NHEJ events occurs precisely with no alteration to joined sequences. Previously, we reported that over-expression of progerin correlated with increased NHEJ relative to HR. We now report on progerin's impact on the nature of DNA end-joining. We used a model system involving a DNA end-joining reporter substrate integrated into the genome of cultured thymidine kinase-deficient mouse fibroblasts. Some cells were engineered to express progerin. Two closely spaced DSBs were induced in the integrated substrate through expression of endonuclease I-SceI, and DSB repair events were recovered through selection for thymidine kinase function. DNA sequencing revealed that progerin expression correlated with a significant shift away from precise end-joining between the two I-SceI sites and toward imprecise end-joining. Additional experiments revealed that progerin did not reduce HR fidelity. Our work suggests that progerin suppresses interactions between complementary sequences at DNA termini, thereby shifting DSB repair toward low-fidelity DNA end-joining and perhaps contributing to accelerated and normal aging through compromised genome stability.
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Affiliation(s)
- Liza A Joudeh
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Alannah J DiCintio
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Madeline R Ries
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Andrew S Gasperson
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Kennedy E Griffin
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Victoria P Robbins
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Makenzie Bonner
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Sarah Nolan
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Emma Black
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Alan S Waldman
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA.
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14
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Cullati SN, Zhang E, Shan Y, Guillen RX, Chen JS, Navarrete-Perea J, Elmore ZC, Ren L, Gygi SP, Gould KL. Fission yeast CK1 promotes DNA double-strand break repair through both homologous recombination and non-homologous end joining. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.27.538600. [PMID: 37162912 PMCID: PMC10168346 DOI: 10.1101/2023.04.27.538600] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/11/2023]
Abstract
The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells activated the DNA damage checkpoint due to persistent double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and non-homologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. In order to understand how Hhp1 and Hhp2 promote DSB repair, we identified new substrates using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 that is important for DSB repair. Our data suggest that Hhp1 and Hhp2 facilitate DSB repair by phosphorylating multiple substrates, including Arp8.
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Affiliation(s)
- Sierra N. Cullati
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Eric Zhang
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
- Current address: Columbia University Medical Center, New York, NY, USA
| | - Yufan Shan
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Rodrigo X. Guillen
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Jun-Song Chen
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
| | | | - Zachary C. Elmore
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
- Current address: Department of Surgery, Duke University School of Medicine, Durham, NC, USA
| | - Liping Ren
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Steven P. Gygi
- Department of Cell Biology, Harvard Medical School, Boston, MA, USA
| | - Kathleen L. Gould
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
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15
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Yu N, Qin H, Zhang F, Liu T, Cao K, Yang Y, Chen Y, Cai J. The role and mechanism of long non-coding RNAs in homologous recombination repair of radiation-induced DNA damage. J Gene Med 2023; 25:e3470. [PMID: 36537017 DOI: 10.1002/jgm.3470] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2022] [Revised: 11/29/2022] [Accepted: 12/04/2022] [Indexed: 12/24/2022] Open
Abstract
DNA double-strand breaks can seriously damage the genetic information that organisms depend on for survival and reproduction. Therefore, cells require a robust DNA damage response mechanism to repair the damaged DNA. Homologous recombination (HR) allows error-free repair, which is key to maintaining genomic integrity. Long non-coding RNAs (lncRNAs) are RNA molecules that are longer than 200 nucleotides. In recent years, a number of studies have found that lncRNAs can act as regulators of gene expression and DNA damage response mechanisms, including HR repair. Moreover, they have significant effects on the occurrence, development, invasion and metastasis of tumor cells, as well as the sensitivity of tumors to radiotherapy and chemotherapy. These studies have therefore begun to expose the great potential of lncRNAs for clinical applications. In this review, we focus on the regulatory roles of lncRNAs in HR repair.
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Affiliation(s)
- Nanxi Yu
- School of Public Health and Management, Wenzhou Medical University, University Town, Wenzhou, China.,South Zhejiang Institute of Radiation Medicine and Nuclear Technology, Wenzhou, China
| | - Hongran Qin
- Department of Nuclear Radiation, Shanghai Pulmonary Hospital,School of Medicine, Tongji University, Shanghai, China
| | - Fangxiao Zhang
- School of Public Health and Management, Wenzhou Medical University, University Town, Wenzhou, China
| | - Tingting Liu
- Department of Radiation Medicine, Faculty of Naval Medicine, Naval Medical University, Shanghai, China
| | - Kun Cao
- Department of Radiation Medicine, Faculty of Naval Medicine, Naval Medical University, Shanghai, China
| | - Yanyong Yang
- Department of Radiation Medicine, Faculty of Naval Medicine, Naval Medical University, Shanghai, China
| | - Yuanyuan Chen
- South Zhejiang Institute of Radiation Medicine and Nuclear Technology, Wenzhou, China.,Department of Radiation Medicine, Faculty of Naval Medicine, Naval Medical University, Shanghai, China
| | - Jianming Cai
- School of Public Health and Management, Wenzhou Medical University, University Town, Wenzhou, China.,South Zhejiang Institute of Radiation Medicine and Nuclear Technology, Wenzhou, China
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16
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A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/Cas13/Cas14 Proteins Utilized for Genome Engineering. Mol Biotechnol 2023; 65:311-325. [PMID: 36163606 PMCID: PMC9512960 DOI: 10.1007/s12033-022-00567-0] [Citation(s) in RCA: 94] [Impact Index Per Article: 47.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Accepted: 09/15/2022] [Indexed: 11/17/2022]
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system has altered life science research offering enormous options in manipulating, detecting, imaging, and annotating specific DNA or RNA sequences of diverse organisms. This system incorporates fragments of foreign DNA (spacers) into CRISPR cassettes, which are further transcribed into the CRISPR arrays and then processed to make guide RNA (gRNA). The CRISPR arrays are genes that encode Cas proteins. Cas proteins provide the enzymatic machinery required for acquiring new spacers targeting invading elements. Due to programmable sequence specificity, numerous Cas proteins such as Cas9, Cas12, Cas13, and Cas14 have been exploited to develop new tools for genome engineering. Cas variants stimulated genetic research and propelled the CRISPR/Cas tool for manipulating and editing nucleic acid sequences of living cells of diverse organisms. This review aims to provide detail on two classes (class 1 and 2) of the CRISPR/Cas system, and the mechanisms of all Cas proteins, including Cas12, Cas13, and Cas14 discovered so far. In addition, we also discuss the pros and cons and recent applications of various Cas proteins in diverse fields, including those used to detect viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This review enables the researcher to gain knowledge on various Cas proteins and their applications, which have the potential to be used in next-generation precise genome engineering.
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17
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Ping X, Stark JM. O-GlcNAc transferase is important for homology-directed repair. DNA Repair (Amst) 2022; 119:103394. [PMID: 36095925 PMCID: PMC9884008 DOI: 10.1016/j.dnarep.2022.103394] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2022] [Revised: 08/11/2022] [Accepted: 09/01/2022] [Indexed: 01/31/2023]
Abstract
O-Linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) to serine or threonine residues is a reversible and dynamic post-translational modification. O-GlcNAc transferase (OGT) is the only enzyme for O-GlcNAcylation, and is a potential cancer therapeutic target in combination with clastogenic (i.e., chromosomal breaking) therapeutics. Thus, we sought to examine the influence of O-GlcNAcylation on chromosomal break repair. Using a set of DNA double strand break (DSB) reporter assays, we found that the depletion of OGT, and its inhibition with a small molecule each caused a reduction in repair pathways that involve use of homology: RAD51-dependent homology-directed repair (HDR), and single strand annealing. In contrast, such OGT disruption did not obviously affect chromosomal break end joining, and furthermore caused an increase in homology-directed gene targeting. Such disruption in OGT also caused a reduction in clonogenic survival, as well as modifications to cell cycle profiles, particularly an increase in G1-phase cells. We also examined intermediate steps of HDR, finding no obvious effects on an assay for DSB end resection, nor for RAD51 recruitment into ionizing radiation induced foci (IRIF) in proliferating cells. However, we also found that the influence of OGT on HDR and homology-directed gene targeting were dependent on RAD52, and that OGT is important for RAD52 IRIF in proliferating cells. Thus, we suggest that OGT is important for regulation of HDR that is partially linked to RAD52 function.
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Affiliation(s)
- Xiaoli Ping
- Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
| | - Jeremy M. Stark
- Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA,Correspondence should be addressed to J.M.S:, Phone: 626-218-6346, Fax: 626-301-8892,
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18
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Hassan MM, Yuan G, Liu Y, Alam M, Eckert CA, Tuskan GA, Golz JF, Yang X. Precision genome editing in plants using gene targeting and prime editing: existing and emerging strategies. Biotechnol J 2022; 17:e2100673. [PMID: 35766313 DOI: 10.1002/biot.202100673] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Revised: 06/16/2022] [Accepted: 06/22/2022] [Indexed: 11/08/2022]
Abstract
Precise modification of plant genomes, such as seamless insertion, deletion, or replacement of DNA sequences at a predefined site, is a challenging task. Gene targeting (GT) and prime editing are currently the best approaches for this purpose. However, these techniques are inefficient in plants, which limits their applications for crop breeding programs. Recently, substantial developments have been made to improve the efficiency of these techniques in plants. Several strategies, such as RNA donor templating, chemically modified donor DNA template, and tandem-repeat homology-directed repair, are aimed at improving GT. Additionally, improved prime editing gRNA design, use of engineered reverse transcriptase enzymes, and splitting prime editing components have improved the efficacy of prime editing in plants. These emerging strategies and existing technologies are reviewed along with various perspectives on their future improvement and the development of robust precision genome editing technologies for plants.
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Affiliation(s)
- Md Mahmudul Hassan
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
- The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
- Department of Genetics and Plant Breeding, Patuakhali Science and Technology University, Dumki, Patuakhali, 8602, Bangladesh
| | - Guoliang Yuan
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
- The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
| | - Yang Liu
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
| | - Mobashwer Alam
- Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Nambour, Queensland, Australia
| | - Carrie A Eckert
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
- The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
| | - Gerald A Tuskan
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
- The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
| | - John F Golz
- School of Biosciences, University of Melbourne, Royal Parade, Parkville, Victoria, 3010, Australia
| | - Xiaohan Yang
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
- The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, USA
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19
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Liu Q, Liu P, Ji T, Zheng L, Shen C, Ran S, Liu J, Zhao Y, Niu Y, Wang T, Dong J. The histone methyltransferase SUVR2 promotes DSB repair via chromatin remodeling and liquid-liquid phase separation. MOLECULAR PLANT 2022; 15:1157-1175. [PMID: 35610973 DOI: 10.1016/j.molp.2022.05.007] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/12/2022] [Revised: 05/15/2022] [Accepted: 05/18/2022] [Indexed: 06/15/2023]
Abstract
Maintaining genomic integrity and stability is particularly important for stem cells, which are at the top of the cell lineage origin. Here, we discovered that the plant-specific histone methyltransferase SUVR2 maintains the genome integrity of the root tip stem cells through chromatin remodeling and liquid-liquid phase separation (LLPS) when facing DNA double-strand breaks (DSBs). The histone methyltransferase SUVR2 (MtSUVR2) has histone methyltransferase activity and catalyzes the conversion of histone H3 lysine 9 monomethylation (H3K9me1) to H3K9me2/3 in vitro and in Medicago truncatula. Under DNA damage, the proportion of heterochromatin decreased and the level of DSB damage marker γ-H2AX increased in suvr2 mutants, indicating that MtSUVR2 promotes the compaction of the chromatin structure through H3K9 methylation modification to protect DNA from damage. Interestingly, MtSUVR2 was induced by DSBs to phase separate and form droplets to localize at the damage sites, and this was confirmed by immunofluorescence and fluorescence recovery after photobleaching experiments. The IDR1 and low-complexity domain regions of MtSUVR2 determined its phase separation in the nucleus, whereas the IDR2 region determined the interaction with the homologous recombinase MtRAD51. Furthermore, we found that MtSUVR2 drove the phase separation of MtRAD51 to form "DNA repair bodies," which could enhance the stability of MtRAD51 proteins to facilitate error-free homologous recombination repair of stem cells. Taken together, our study reveals that chromatin remodeling-associated proteins participate in DNA repair through LLPS.
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Affiliation(s)
- Qianwen Liu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Peng Liu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Tuo Ji
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Lihua Zheng
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Chen Shen
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Shasha Ran
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Jinling Liu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Yafei Zhao
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Yiding Niu
- Key Laboratory of Forage and Endemic Crop Biology, Ministry of Education, School of Life Sciences, Inner Mongolia University, Hohhot, China
| | - Tao Wang
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
| | - Jiangli Dong
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
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20
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Xiao H, Li F, Mladenov E, Soni A, Mladenova V, Pan B, Dueva R, Stuschke M, Timmermann B, Iliakis G. Increased Resection at DSBs in G2-Phase Is a Unique Phenotype Associated with DNA-PKcs Defects That Is Not Shared by Other Factors of c-NHEJ. Cells 2022; 11:cells11132099. [PMID: 35805183 PMCID: PMC9265841 DOI: 10.3390/cells11132099] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 06/28/2022] [Accepted: 06/30/2022] [Indexed: 01/27/2023] Open
Abstract
The load of DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by different doses of ionizing radiation (IR) is a key determinant of DSB repair pathway choice, with homologous recombination (HR) and ATR substantially gaining ground at doses below 0.5 Gy. Increased resection and HR engagement with decreasing DSB-load generate a conundrum in a classical non-homologous end-joining (c-NHEJ)-dominated cell and suggest a mechanism adaptively facilitating resection. We report that ablation of DNA-PKcs causes hyper-resection, implicating DNA-PK in the underpinning mechanism. However, hyper-resection in DNA-PKcs-deficient cells can also be an indirect consequence of their c-NHEJ defect. Here, we report that all tested DNA-PKcs mutants show hyper-resection, while mutants with defects in all other factors of c-NHEJ fail to do so. This result rules out the model of c-NHEJ versus HR competition and the passive shift from c-NHEJ to HR as the causes of the increased resection and suggests the integration of DNA-PKcs into resection regulation. We develop a model, compatible with the results of others, which integrates DNA-PKcs into resection regulation and HR for a subset of DSBs. For these DSBs, we propose that the kinase remains at the break site, rather than the commonly assumed autophosphorylation-mediated removal from DNA ends.
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Affiliation(s)
- Huaping Xiao
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (H.X.); (F.L.); (E.M.); (A.S.); (V.M.); (B.P.); (R.D.)
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
| | - Fanghua Li
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (H.X.); (F.L.); (E.M.); (A.S.); (V.M.); (B.P.); (R.D.)
- Department of Particle Therapy, University Hospital Essen, West German Proton Therapy Centre Essen (WPE), West German Cancer Center (WTZ), German Cancer Consortium (DKTK), 45147 Essen, Germany;
| | - Emil Mladenov
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (H.X.); (F.L.); (E.M.); (A.S.); (V.M.); (B.P.); (R.D.)
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
| | - Aashish Soni
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (H.X.); (F.L.); (E.M.); (A.S.); (V.M.); (B.P.); (R.D.)
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
| | - Veronika Mladenova
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (H.X.); (F.L.); (E.M.); (A.S.); (V.M.); (B.P.); (R.D.)
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
| | - Bing Pan
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (H.X.); (F.L.); (E.M.); (A.S.); (V.M.); (B.P.); (R.D.)
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
| | - Rositsa Dueva
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (H.X.); (F.L.); (E.M.); (A.S.); (V.M.); (B.P.); (R.D.)
- Institute of Physiology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany
| | - Martin Stuschke
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
- German Cancer Consortium (DKTK), Partner Site University Hospital Essen, German Cancer Research Center (DKFZ), 45147 Essen, Germany
| | - Beate Timmermann
- Department of Particle Therapy, University Hospital Essen, West German Proton Therapy Centre Essen (WPE), West German Cancer Center (WTZ), German Cancer Consortium (DKTK), 45147 Essen, Germany;
- German Cancer Consortium (DKTK), Partner Site University Hospital Essen, German Cancer Research Center (DKFZ), 45147 Essen, Germany
| | - George Iliakis
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (H.X.); (F.L.); (E.M.); (A.S.); (V.M.); (B.P.); (R.D.)
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
- Correspondence: ; Tel.: +49-201-723-4152
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21
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Chen Y, Banie L, Breyer BN, Tan Y, Wang Z, Zhou F, Wang G, Lin G, Liu J, Qi LS, Lue TF. Enhanced Myogenesis by Silencing Myostatin with Nonviral Delivery of dCas9 Ribonucleoprotein Complex. CRISPR J 2022; 5:598-608. [PMID: 35758824 DOI: 10.1089/crispr.2022.0009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to restore urethral striated and pelvic floor muscles represents a valuable therapeutic approach. We aim to determine the CRISPR interference-mediated gene silencing effect of the nonviral delivery of nuclease-deactivated dCas9 ribonucleoprotein (RNP) complex on muscle regeneration at the cellular and molecular level. We designed four myostatin (MSTN)-targeting sgRNAs and transfected them into rat myoblast L6 cells together with the dCas9 protein. Myogenesis assay and immunofluorescence staining were performed to evaluate muscle differentiation, while CCK8 assay, cell cycle assay, and 5-ethynyl-2'-deoxyuridine staining were used to measure muscle proliferation. Reverse transcription-polymerase chain reaction and Western blotting were also performed to examine cellular signaling. Myogenic factors (including myosin heavy chain, MSTN, myocardin, and serum response factor) increased significantly after day 5 during myogenesis. MSTN was efficiently silenced after transfecting the dCas9 RNP complex, which significantly promoted more myotube formation and a higher fusion index for L6 cells. In cellular signaling, MSTN repression enhanced the expression of MyoG and MyoD, phosphorylation of Smad2, and the activity of Wnt1/GSK-3β/β-catenin pathway. Moreover, MSTN repression accelerated L6 cell growth with a higher cell proliferation index as well as a higher expression of cyclin D1 and cyclin E. Nonviral delivery of the dCas9 RNP complex significantly promoted myoblast differentiation and proliferation, providing a promising approach to improve muscle regeneration for SUI and PFD. Further characterization and validation of this approach in vivo are needed.
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Affiliation(s)
- Yinwei Chen
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA.,Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Lia Banie
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Benjamin N Breyer
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Yan Tan
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Zhao Wang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Feng Zhou
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Guifang Wang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Guiting Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Jihong Liu
- Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Lei S Qi
- Department of Bioengineering, Stanford University, Stanford, California, USA.,ChEM-H, Stanford University, Stanford, California, USA
| | - Tom F Lue
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
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22
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Chen H, Neubauer M, Wang JP. Enhancing HR Frequency for Precise Genome Editing in Plants. FRONTIERS IN PLANT SCIENCE 2022; 13:883421. [PMID: 35592579 PMCID: PMC9113527 DOI: 10.3389/fpls.2022.883421] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/25/2022] [Accepted: 03/29/2022] [Indexed: 06/15/2023]
Abstract
Gene-editing tools, such as Zinc-fingers, TALENs, and CRISPR-Cas, have fostered a new frontier in the genetic improvement of plants across the tree of life. In eukaryotes, genome editing occurs primarily through two DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the primary mechanism in higher plants, but it is unpredictable and often results in undesired mutations, frameshift insertions, and deletions. Homology-directed repair (HDR), which proceeds through HR, is typically the preferred editing method by genetic engineers. HR-mediated gene editing can enable error-free editing by incorporating a sequence provided by a donor template. However, the low frequency of native HR in plants is a barrier to attaining efficient plant genome engineering. This review summarizes various strategies implemented to increase the frequency of HDR in plant cells. Such strategies include methods for targeting double-strand DNA breaks, optimizing donor sequences, altering plant DNA repair machinery, and environmental factors shown to influence HR frequency in plants. Through the use and further refinement of these methods, HR-based gene editing may one day be commonplace in plants, as it is in other systems.
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Affiliation(s)
- Hao Chen
- Department of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, United States
- College of Forestry, Shandong Agricultural University, Tai’an, China
| | - Matthew Neubauer
- Department of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, United States
| | - Jack P. Wang
- Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, NC, United States
- State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China
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23
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Foe VE. Does the Pachytene Checkpoint, a Feature of Meiosis, Filter Out Mistakes in Double-Strand DNA Break Repair and as a side-Effect Strongly Promote Adaptive Speciation? Integr Org Biol 2022; 4:obac008. [PMID: 36827645 PMCID: PMC8998493 DOI: 10.1093/iob/obac008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
This essay aims to explain two biological puzzles: why eukaryotic transcription units are composed of short segments of coding DNA interspersed with long stretches of non-coding (intron) DNA, and the near ubiquity of sexual reproduction. As is well known, alternative splicing of its coding sequences enables one transcription unit to produce multiple variants of each encoded protein. Additionally, padding transcription units with non-coding DNA (often many thousands of base pairs long) provides a readily evolvable way to set how soon in a cell cycle the various mRNAs will begin being expressed and the total amount of mRNA that each transcription unit can make during a cell cycle. This regulation complements control via the transcriptional promoter and facilitates the creation of complex eukaryotic cell types, tissues, and organisms. However, it also makes eukaryotes exceedingly vulnerable to double-strand DNA breaks, which end-joining break repair pathways can repair incorrectly. Transcription units cover such a large fraction of the genome that any mis-repair producing a reorganized chromosome has a high probability of destroying a gene. During meiosis, the synaptonemal complex aligns homologous chromosome pairs and the pachytene checkpoint detects, selectively arrests, and in many organisms actively destroys gamete-producing cells with chromosomes that cannot adequately synapse; this creates a filter favoring transmission to the next generation of chromosomes that retain the parental organization, while selectively culling those with interrupted transcription units. This same meiotic checkpoint, reacting to accidental chromosomal reorganizations inflicted by error-prone break repair, can, as a side effect, provide a mechanism for the formation of new species in sympatry. It has been a long-standing puzzle how something as seemingly maladaptive as hybrid sterility between such new species can arise. I suggest that this paradox is resolved by understanding the adaptive importance of the pachytene checkpoint, as outlined above.
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24
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Elmas E, Saljoughian N, de Souza Fernandes Pereira M, Tullius BP, Sorathia K, Nakkula RJ, Lee DA, Naeimi Kararoudi M. CRISPR Gene Editing of Human Primary NK and T Cells for Cancer Immunotherapy. Front Oncol 2022; 12:834002. [PMID: 35449580 PMCID: PMC9016158 DOI: 10.3389/fonc.2022.834002] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2021] [Accepted: 03/07/2022] [Indexed: 11/13/2022] Open
Abstract
Antitumor activity of immune cells such as T cells and NK cells has made them auspicious therapeutic regimens for adaptive cancer immunotherapy. Enhancing their cytotoxic effects against malignancies and overcoming their suppression in tumor microenvironment (TME) may improve their efficacy to treat cancers. Clustered, regularly interspaced short palindromic repeats (CRISPR) genome editing has become one of the most popular tools to enhance immune cell antitumor activity. In this review we highlight applications and practicability of CRISPR/Cas9 gene editing and engineering strategies for cancer immunotherapy. In addition, we have reviewed several approaches to study CRISPR off-target effects.
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Affiliation(s)
- Ezgi Elmas
- Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, United States
- Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States
| | - Noushin Saljoughian
- Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States
- CRISPR/Gene Editing Core, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States
| | - Marcelo de Souza Fernandes Pereira
- Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States
| | - Brian P. Tullius
- Pediatric Cellular Therapy, AdventHealth for Children, Orlando, FL, United States
| | - Kinnari Sorathia
- Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States
| | - Robin J. Nakkula
- Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States
| | - Dean A. Lee
- Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States
- Department of Pediatrics, The Ohio State University, Columbus, OH, United States
| | - Meisam Naeimi Kararoudi
- Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States
- CRISPR/Gene Editing Core, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States
- Department of Pediatrics, The Ohio State University, Columbus, OH, United States
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25
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Wang S, Li Y, Zhong L, Wu K, Zhang R, Kang T, Wu S, Wu Y. Efficient gene editing through an intronic selection marker in cells. Cell Mol Life Sci 2022; 79:111. [PMID: 35098362 PMCID: PMC8801403 DOI: 10.1007/s00018-022-04152-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2021] [Revised: 01/10/2022] [Accepted: 01/14/2022] [Indexed: 02/05/2023]
Abstract
BACKGROUND Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. However, despite the great importance and universality of gene editing, the efficiency of homology-directed DNA repair (HDR) is too low, and base editors (BEs) cannot accomplish desired indel editing tasks. RESULTS AND DISCUSSION Our group has improved HDR gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves a DsRed positive rate as high as 87.5% after two rounds of fluorescence-activated cell sorter (FACS) selection without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and efficiency, and our data suggest that GEIS has the potential to edit approximately 97% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.
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Affiliation(s)
- Shang Wang
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Department of Experimental Research, Sun Yat-Sen University Cancer Center, Guangzhou, 510060, China
- Institute of Urology, The Third Affiliated Hospital of Shenzhen University, Shenzhen, 518000, China
| | - Yuqing Li
- Institute of Urology, The Third Affiliated Hospital of Shenzhen University, Shenzhen, 518000, China
- Teaching Center of Shenzhen Luohu Hospital, Shantou University Medical College, Shantou, 515000, China
| | - Li Zhong
- Center of Digestive Diseases, The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen, 518107, China
- Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen, 518107, China
| | - Kai Wu
- Institute of Urology, The Third Affiliated Hospital of Shenzhen University, Shenzhen, 518000, China
| | - Ruhua Zhang
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Department of Experimental Research, Sun Yat-Sen University Cancer Center, Guangzhou, 510060, China
| | - Tiebang Kang
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Department of Experimental Research, Sun Yat-Sen University Cancer Center, Guangzhou, 510060, China
| | - Song Wu
- Institute of Urology, The Third Affiliated Hospital of Shenzhen University, Shenzhen, 518000, China.
- Teaching Center of Shenzhen Luohu Hospital, Shantou University Medical College, Shantou, 515000, China.
- Department of Urology, South China Hospital of Shenzhen University, Shenzhen, 518000, China.
| | - Yuanzhong Wu
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Department of Experimental Research, Sun Yat-Sen University Cancer Center, Guangzhou, 510060, China.
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Abstract
Actin is a highly conserved protein in mammals. The actin dynamics is regulated by actin-binding proteins and actin-related proteins. Nuclear actin and these regulatory proteins participate in multiple nuclear processes, including chromosome architecture organization, chromatin remodeling, transcription machinery regulation, and DNA repair. It is well known that the dysfunctions of these processes contribute to the development of cancer. Moreover, emerging evidence has shown that the deregulated actin dynamics is also related to cancer. This chapter discusses how the deregulation of nuclear actin dynamics contributes to tumorigenesis via such various nuclear events.
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Affiliation(s)
- Yuanjian Huang
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China
| | - Shengzhe Zhang
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Jae-Il Park
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
- Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center and Health Science Center, Houston, TX, USA.
- Program in Genetics and Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
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27
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Vu TV, Das S, Nguyen CC, Kim J, Kim JY. Single-strand annealing: Molecular mechanisms and potential applications in CRISPR-Cas-based precision genome editing. Biotechnol J 2021; 17:e2100413. [PMID: 34846104 DOI: 10.1002/biot.202100413] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Revised: 11/22/2021] [Accepted: 11/29/2021] [Indexed: 12/24/2022]
Abstract
BACKGROUND Spontaneous double-stranded DNA breaks (DSBs) frequently occur within the genome of all living organisms and must be well repaired for survival. Recently, more important roles of the DSB repair pathways that were previously thought to be minor pathways, such as single-strand annealing (SSA), have been shown. Nevertheless, the biochemical mechanisms and applications of the SSA pathway in genome editing have not been updated. PURPOSE AND SCOPE Understanding the molecular mechanism of SSA is important to design potential applications in gene editing. This review provides insights into the recent progress of SSA studies and establishes a model for their potential applications in precision genome editing. SUMMARY AND CONCLUSION The SSA mechanism involved in DNA DSB repair appears to be activated by a complex signaling cascade starting with broken end sensing and 5'-3' resection to reveal homologous repeats on the 3' ssDNA overhangs that flank the DSB. Annealing the repeats would help to amend the discontinuous ends and restore the intact genome, resulting in the missing of one repeat and the intervening sequence between the repeats. We proposed a model for CRISPR-Cas-based precision insertion or replacement of DNA fragments to take advantage of the characteristics. The proposed model can add a tool to extend the choice for precision gene editing. Nevertheless, the model needs to be experimentally validated and optimized with SSA-favorable conditions for practical applications.
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Affiliation(s)
- Tien Van Vu
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea.,National Key Laboratory for Plant Cell Biotechnology, Agricultural Genetics Institute, Bac Tu Liem, Hanoi, Vietnam
| | - Swati Das
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea
| | - Cam Chau Nguyen
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea
| | - Jihae Kim
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea
| | - Jae-Yean Kim
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea.,Division of Life Science, Gyeongsang National University, Jinju, Republic of Korea
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28
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ZGRF1 promotes end resection of DNA homologous recombination via forming complex with BRCA1/EXO1. Cell Death Discov 2021; 7:260. [PMID: 34552057 PMCID: PMC8458317 DOI: 10.1038/s41420-021-00633-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2021] [Revised: 08/16/2021] [Accepted: 08/26/2021] [Indexed: 11/08/2022] Open
Abstract
To maintain genomic stability, the mammalian cells has evolved a coordinated response to DNA damage, including activation of DNA repair and cell cycle checkpoint processes. Exonuclease 1 (EXO1)-dependent excision of DNA ends is important for the initiation of homologous recombination (HR) repair of DNA breaks, which is thought to play a key role in activating the ATR-CHK1 pathway to induce G2/M cell cycle arrest. But the mechanism is still not fully understood. Here, we report that ZGRF1 forms complexes with EXO1 as well as other repair proteins and promotes DNA repair through HR. ZGRF1 is recruited to DNA damage sites in a MDC1-RNF8-BRCA1 dependent manner. Furthermore, ZGRF1 is important for the recruitment of RPA2 to DNA damage sites and the following ATR-CHK1 mediated G2/M checkpoint in response to irradiation. ZGRF1 null cells show increased sensitivity to many DNA-damaging agents, especially PARPi and irradiation. Collectively,our findings identify ZGRF1 as a novel regulator of DNA end resection and G2/M checkpoint. ZGRF1 is a potential target of radiation and PARPi cancer therapy.
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29
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Nickoloff JA, Sharma N, Allen CP, Taylor L, Allen SJ, Jaiswal AS, Hromas R. Roles of homologous recombination in response to ionizing radiation-induced DNA damage. Int J Radiat Biol 2021; 99:903-914. [PMID: 34283012 PMCID: PMC9629169 DOI: 10.1080/09553002.2021.1956001] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 03/04/2021] [Accepted: 07/05/2021] [Indexed: 02/06/2023]
Abstract
PURPOSE Ionizing radiation induces a vast array of DNA lesions including base damage, and single- and double-strand breaks (SSB, DSB). DSBs are among the most cytotoxic lesions, and mis-repair causes small- and large-scale genome alterations that can contribute to carcinogenesis. Indeed, ionizing radiation is a 'complete' carcinogen. DSBs arise immediately after irradiation, termed 'frank DSBs,' as well as several hours later in a replication-dependent manner, termed 'secondary' or 'replication-dependent DSBs. DSBs resulting from replication fork collapse are single-ended and thus pose a distinct problem from two-ended, frank DSBs. DSBs are repaired by error-prone nonhomologous end-joining (NHEJ), or generally error-free homologous recombination (HR), each with sub-pathways. Clarifying how these pathways operate in normal and tumor cells is critical to increasing tumor control and minimizing side effects during radiotherapy. CONCLUSIONS The choice between NHEJ and HR is regulated during the cell cycle and by other factors. DSB repair pathways are major contributors to cell survival after ionizing radiation, including tumor-resistance to radiotherapy. Several nucleases are important for HR-mediated repair of replication-dependent DSBs and thus replication fork restart. These include three structure-specific nucleases, the 3' MUS81 nuclease, and two 5' nucleases, EEPD1 and Metnase, as well as three end-resection nucleases, MRE11, EXO1, and DNA2. The three structure-specific nucleases evolved at very different times, suggesting incremental acceleration of replication fork restart to limit toxic HR intermediates and genome instability as genomes increased in size during evolution, including the gain of large numbers of HR-prone repetitive elements. Ionizing radiation also induces delayed effects, observed days to weeks after exposure, including delayed cell death and delayed HR. In this review we highlight the roles of HR in cellular responses to ionizing radiation, and discuss the importance of HR as an exploitable target for cancer radiotherapy.
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Affiliation(s)
- Jac A. Nickoloff
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
| | - Neelam Sharma
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
| | - Christopher P. Allen
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
- Department of Microbiology, Immunology and Pathology, Flow Cytometry and Cell Sorting Facility, Colorado State University, Fort Collins, CO, USA
| | - Lynn Taylor
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
| | - Sage J. Allen
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
| | - Aruna S. Jaiswal
- Division of Hematology and Medical Oncology, Department of Medicine and the Mays Cancer Center, University of Texas Health Science Center, San Antonio, TX, USA
| | - Robert Hromas
- Division of Hematology and Medical Oncology, Department of Medicine and the Mays Cancer Center, University of Texas Health Science Center, San Antonio, TX, USA
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30
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Gillyard T, Davis J. DNA double-strand break repair in cancer: A path to achieving precision medicine. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2021; 364:111-137. [PMID: 34507781 DOI: 10.1016/bs.ircmb.2021.06.003] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The assessment of DNA damage can be a significant diagnostic for precision medicine. DNA double strand break (DSBs) pathways in cancer are the primary targets in a majority of anticancer therapies, yet the molecular vulnerabilities that underlie each tumor can vary widely making the application of precision medicine challenging. Identifying and understanding these interindividual vulnerabilities enables the design of targeted DSB inhibitors along with evolving precision medicine approaches to selectively kill cancer cells with minimal side effects. A major challenge however, is defining exactly how to target unique differences in DSB repair pathway mechanisms. This review comprises a brief overview of the DSB repair mechanisms in cancer and includes results obtained with revolutionary advances such as CRISPR/Cas9 and machine learning/artificial intelligence, which are rapidly advancing not only our understanding of determinants of DSB repair choice, but also how it can be used to advance precision medicine. Scientific innovation in the methods used to diagnose and treat cancer is converging with advances in basic science and translational research. This revolution will continue to be a critical driver of precision medicine that will enable precise targeting of unique individual mechanisms. This review aims to lay the foundation for achieving this goal.
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Affiliation(s)
- Taneisha Gillyard
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, TN, United States
| | - Jamaine Davis
- Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, TN, United States.
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31
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Potential of helper-dependent Adenoviral vectors in CRISPR-cas9-mediated lung gene therapy. Cell Biosci 2021; 11:145. [PMID: 34301308 PMCID: PMC8305863 DOI: 10.1186/s13578-021-00662-w] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2021] [Accepted: 07/19/2021] [Indexed: 12/20/2022] Open
Abstract
Since CRISPR/Cas9 was harnessed to edit DNA, the field of gene therapy has witnessed great advances in gene editing. New avenues were created for the treatment of diseases such as Cystic Fibrosis (CF). CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Despite the success of gene editing with the CRISPR/Cas9 in vitro, challenges still exist when using CRISPR/Cas9 in vivo to cure CF lung disease. The delivery of CRISPR/Cas9 into lungs, as well as the difficulty to achieve the efficiency required for clinical efficacy, has brought forth new challenges. Viral and non-viral vectors have been shown to deliver DNA successfully in vivo, but the sustained expression of CFTR was not adequate. Before the introduction of Helper-Dependent Adenoviral vectors (HD-Ad), clinical trials of treating pulmonary genetic diseases with first-generation viral vectors have shown limited efficacy. With the advantages of larger capacity and lower immunogenicity of HD-Ad, together with the versatility of the CRISPR/Cas9 system, delivering CRISPR/Cas9 to the airway with HD-Ad for lung gene therapy shows great potential. In this review, we discuss the status of the application of CRISPR/Cas9 in CF gene therapy, the existing challenges in the field, as well as new hurdles introduced by the presence of CRISPR/Cas9 in the lungs. Through the analysis of these challenges, we present the potential of CRISPR/Cas9-mediated lung gene therapy using HD-Ad vectors with Cystic Fibrosis lung disease as a model of therapy.
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32
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PUMA facilitates EMI1-promoted cytoplasmic Rad51 ubiquitination and inhibits DNA repair in stem and progenitor cells. Signal Transduct Target Ther 2021; 6:129. [PMID: 33785736 PMCID: PMC8009889 DOI: 10.1038/s41392-021-00510-w] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2020] [Revised: 11/20/2020] [Accepted: 01/18/2021] [Indexed: 02/07/2023] Open
Abstract
Maintenance of genetic stability via proper DNA repair in stem and progenitor cells is essential for the tissue repair and regeneration, while preventing cell transformation after damage. Loss of PUMA dramatically increases the survival of mice after exposure to a lethal dose of ionizing radiation (IR), while without promoting tumorigenesis in the long-term survivors. This finding suggests that PUMA (p53 upregulated modulator of apoptosis) may have a function other than regulates apoptosis. Here, we identify a novel role of PUMA in regulation of DNA repair in embryonic or induced pluripotent stem cells (PSCs) and immortalized hematopoietic progenitor cells (HPCs) after IR. We found that PUMA-deficient PSCs and HPCs exhibited a significant higher double-strand break (DSB) DNA repair activity via Rad51-mediated homologous recombination (HR). This is because PUMA can be associated with early mitotic inhibitor 1 (EMI1) and Rad51 in the cytoplasm to facilitate EMI1-mediated cytoplasmic Rad51 ubiquitination and degradation, thereby inhibiting Rad51 nuclear translocation and HR DNA repair. Our data demonstrate that PUMA acts as a repressor for DSB DNA repair and thus offers a new rationale for therapeutic targeting of PUMA in regenerative cells in the context of DNA damage.
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Mitochondrial genome stability in human: understanding the role of DNA repair pathways. Biochem J 2021; 478:1179-1197. [DOI: 10.1042/bcj20200920] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2020] [Revised: 02/15/2021] [Accepted: 02/17/2021] [Indexed: 11/17/2022]
Abstract
Mitochondria are semiautonomous organelles in eukaryotic cells and possess their own genome that replicates independently. Mitochondria play a major role in oxidative phosphorylation due to which its genome is frequently exposed to oxidative stress. Factors including ionizing radiation, radiomimetic drugs and replication fork stalling can also result in different types of mutations in mitochondrial DNA (mtDNA) leading to genome fragility. Mitochondria from myopathies, dystonia, cancer patient samples show frequent mtDNA mutations such as point mutations, insertions and large-scale deletions that could account for mitochondria-associated disease pathogenesis. The mechanism by which such mutations arise following exposure to various DNA-damaging agents is not well understood. One of the well-studied repair pathways in mitochondria is base excision repair. Other repair pathways such as mismatch repair, homologous recombination and microhomology-mediated end joining have also been reported. Interestingly, nucleotide excision repair and classical nonhomologous DNA end joining are not detected in mitochondria. In this review, we summarize the potential causes of mitochondrial genome fragility, their implications as well as various DNA repair pathways that operate in mitochondria.
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Morales ME, Kaul T, Walker J, Everett C, White T, Deininger P. Altered DNA repair creates novel Alu/Alu repeat-mediated deletions. Hum Mutat 2021; 42:600-613. [PMID: 33675284 PMCID: PMC8068675 DOI: 10.1002/humu.24193] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Revised: 01/25/2021] [Accepted: 02/24/2021] [Indexed: 12/21/2022]
Abstract
Alu elements are the most abundant source of nonallelic homology that influences genetic instability in the human genome. When there is a DNA double-stranded break, the Alu element's high copy number, moderate length and distance and mismatch between elements uniquely influence recombination processes. We utilize a reporter-gene assay to show the complex influence of Alu mismatches on Alu-related repeat-mediated deletions (RMDs). The Alu/Alu heteroduplex intermediate can result in a nonallelic homologous recombination (HR). Alternatively, the heteroduplex can result in various DNA breaks around the Alu elements caused by competing nucleases. These breaks can undergo Alt-nonhomologous end joining to cause deletions focused around the Alu elements. Formation of these heteroduplex intermediates is largely RAD52 dependent. Cells with low ERCC1 levels utilize more of these alternatives resolutions, while cells with MSH2 defects tend to have more RMDs with a specific increase in the HR events. Therefore, Alu elements are expected to create different forms of deletions in various cancers depending on a number of these DNA repair defects.
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Affiliation(s)
- Maria E Morales
- Tulane Cancer Center, Tulane University, New Orleans, Louisiana, USA
| | - Tiffany Kaul
- Tulane Cancer Center, Tulane University, New Orleans, Louisiana, USA
| | - JaNiece Walker
- Department of Biology, Xavier University, New Orleans, Louisiana, USA
| | - Chelsea Everett
- Tulane Cancer Center, Tulane University, New Orleans, Louisiana, USA
| | - Travis White
- Tulane Cancer Center, Tulane University, New Orleans, Louisiana, USA.,Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Prescott Deininger
- Tulane Cancer Center, Tulane University, New Orleans, Louisiana, USA.,Department of Epidemiology, Tulane University, New Orleans, Louisiana, USA
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Roy U, Kwon Y, Marie L, Symington L, Sung P, Lisby M, Greene EC. The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination. Mol Cell 2021; 81:1043-1057.e8. [PMID: 33421364 PMCID: PMC8262405 DOI: 10.1016/j.molcel.2020.12.019] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2020] [Revised: 11/02/2020] [Accepted: 12/10/2020] [Indexed: 12/29/2022]
Abstract
Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2.
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Affiliation(s)
- Upasana Roy
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA
| | - Youngho Kwon
- Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
| | - Lea Marie
- Department of Microbiology and Immunology, Columbia University, New York, NY 10032, USA
| | - Lorraine Symington
- Department of Microbiology and Immunology, Columbia University, New York, NY 10032, USA
| | - Patrick Sung
- Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
| | - Michael Lisby
- Department of Biology, University of Copenhagen, 2200 Copenhagen N, Denmark; Center for Chromosome Stability, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen N, Denmark
| | - Eric C Greene
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
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Cuarenta A, Kigar SL, Henion IC, Chang L, Bakshi VP, Auger AP. Early life stress during the neonatal period alters social play and Line1 during the juvenile stage of development. Sci Rep 2021; 11:3549. [PMID: 33574362 PMCID: PMC7878767 DOI: 10.1038/s41598-021-82953-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Accepted: 01/25/2021] [Indexed: 12/12/2022] Open
Abstract
Early life stress (ELS) has been shown to have a significant impact on typical brain development and the manifestation of psychological disorders through epigenetic modifications that alter gene expression. Line1, a retrotransposon associated with genetic diversity, has been linked with various psychological disorders that are associated with ELS. Our previous work demonstrated altered Line1 DNA copy number in the neonatal period following stressful experiences; we therefore chose to investigate whether early life stress altered Line1 retrotransposition persists into the juvenile period of development. Our study uses a neonatal predator odor exposure (POE) paradigm to model ELS in rats. We examined Line1 using qPCR to assess Line1 expression levels and DNA copy number in the male and female juvenile amygdala, hippocampus and prefrontal cortex-areas chosen for their association with affective disorders and stress. We report a sex difference in Line1 levels within the juvenile amygdala. We also find that ELS significantly increases Line1 DNA copy number within the juvenile amygdala which correlates with reduced juvenile social play levels, suggesting the possibility that Line1 may influence juvenile social development.
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Affiliation(s)
- Amelia Cuarenta
- Department of Psychology, University of Wisconsin-Madison, Madison, USA
| | - Stacey L Kigar
- Molecular and Cellular Pharmacology Training Program, University of Wisconsin-Madison, Madison, USA
| | - Ian C Henion
- Department of Psychology, University of Wisconsin-Madison, Madison, USA
| | - Liza Chang
- Department of Psychology, University of Wisconsin-Madison, Madison, USA
| | - Vaishali P Bakshi
- Department of Psychiatry, University of Wisconsin-Madison, Madison, USA
| | - Anthony P Auger
- Department of Psychology, University of Wisconsin-Madison, Madison, USA. .,Neuroscience Training Program, University of Wisconsin-Madison, Madison, USA.
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Updates in Paracoccidioides Biology and Genetic Advances in Fungus Manipulation. J Fungi (Basel) 2021; 7:jof7020116. [PMID: 33557381 PMCID: PMC7915485 DOI: 10.3390/jof7020116] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 01/30/2021] [Accepted: 02/02/2021] [Indexed: 12/28/2022] Open
Abstract
The dimorphic fungi of the Paracoccidioides genus are the causative agents of paracoccidioidomycosis (PCM). This disease is endemic in Latin America and primarily affects workers in rural areas. PCM is considered a neglected disease, despite being a disabling disease that has a notable impact on the public health system. Paracoccidioides spp. are thermally dimorphic fungi that present infective mycelia at 25 °C and differentiate into pathogenic yeast forms at 37 °C. This transition involves a series of morphological, structural, and metabolic changes which are essential for their survival inside hosts. As a pathogen, the fungus is subjected to several varieties of stress conditions, including the host immune response, which involves the production of reactive nitrogen and oxygen species, thermal stress due to temperature changes during the transition, pH alterations within phagolysosomes, and hypoxia inside granulomas. Over the years, studies focusing on understanding the establishment and development of PCM have been conducted with several limitations due to the low effectiveness of strategies for the genetic manipulation of Paracoccidioides spp. This review describes the most relevant biological features of Paracoccidioides spp., including aspects of the phylogeny, ecology, stress response, infection, and evasion mechanisms of the fungus. We also discuss the genetic aspects and difficulties of fungal manipulation, and, finally, describe the advances in molecular biology that may be employed in molecular research on this fungus in the future.
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Kumari S, Mukherjee S, Sinha D, Abdisalaam S, Krishnan S, Asaithamby A. Immunomodulatory Effects of Radiotherapy. Int J Mol Sci 2020; 21:E8151. [PMID: 33142765 PMCID: PMC7663574 DOI: 10.3390/ijms21218151] [Citation(s) in RCA: 52] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2020] [Revised: 10/22/2020] [Accepted: 10/26/2020] [Indexed: 02/07/2023] Open
Abstract
Radiation therapy (RT), an integral component of curative treatment for many malignancies, can be administered via an increasing array of techniques. In this review, we summarize the properties and application of different types of RT, specifically, conventional therapy with x-rays, stereotactic body RT, and proton and carbon particle therapies. We highlight how low-linear energy transfer (LET) radiation induces simple DNA lesions that are efficiently repaired by cells, whereas high-LET radiation causes complex DNA lesions that are difficult to repair and that ultimately enhance cancer cell killing. Additionally, we discuss the immunogenicity of radiation-induced tumor death, elucidate the molecular mechanisms by which radiation mounts innate and adaptive immune responses and explore strategies by which we can increase the efficacy of these mechanisms. Understanding the mechanisms by which RT modulates immune signaling and the key players involved in modulating the RT-mediated immune response will help to improve therapeutic efficacy and to identify novel immunomodulatory drugs that will benefit cancer patients undergoing targeted RT.
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Affiliation(s)
- Sharda Kumari
- Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; (S.K.); (D.S.); (S.A.)
| | - Shibani Mukherjee
- Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; (S.K.); (D.S.); (S.A.)
| | - Debapriya Sinha
- Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; (S.K.); (D.S.); (S.A.)
| | - Salim Abdisalaam
- Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; (S.K.); (D.S.); (S.A.)
| | - Sunil Krishnan
- Department of Radiation Oncology, Mayo Clinic Florida, Jacksonville, FL 32224, USA;
| | - Aroumougame Asaithamby
- Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; (S.K.); (D.S.); (S.A.)
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Komari CJ, Guttman AO, Carr SR, Trachtenberg TL, Orloff EA, Haas AV, Patrick AR, Chowdhary S, Waldman BC, Waldman AS. Alteration of genetic recombination and double-strand break repair in human cells by progerin expression. DNA Repair (Amst) 2020; 96:102975. [PMID: 33010688 DOI: 10.1016/j.dnarep.2020.102975] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2020] [Revised: 08/21/2020] [Accepted: 09/04/2020] [Indexed: 01/04/2023]
Abstract
Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare autosomal, dominant genetic condition characterized by many features of accelerated aging. On average, children with HGPS live to about fourteen years of age. The syndrome is commonly caused by a point mutation in the LMNA gene which normally codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The LMNA mutation alters splicing, leading to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at very low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs), suggesting corruption of DNA repair. In this work, we investigated the influence of progerin expression on DSB repair in the human genome at the nucleotide level. We used a model system that involves a reporter DNA substrate inserted in the genome of cultured human cells. A DSB could be induced within the substrate through exogenous expression of endonuclease I-SceI, and DSB repair events occurring via either homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recoverable. Additionally, spontaneous HR events were recoverable in the absence of artificial DSB induction. We compared DSB repair and spontaneous HR in cells overexpressing progerin versus cells expressing no progerin. We report that overexpression of progerin correlated with an increase in DSB repair via NHEJ relative to HR, as well as an increased fraction of HR events occurring via gene conversion. Progerin also engendered an apparent increase in spontaneous HR events, with a highly significant shift toward gene conversion events, and an increase in DNA amplification events. Such influences of progerin on DNA transactions may impact genome stability and contribute to aging.
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Affiliation(s)
- Celina J Komari
- Department of Public Health, University of South Carolina, Columbia, SC 29208, USA
| | - Anne O Guttman
- Department of Exercise Science, University of South Carolina, Columbia, SC 29208, USA
| | - Shelby R Carr
- Department of Exercise Science, University of South Carolina, Columbia, SC 29208, USA
| | - Taylor L Trachtenberg
- Department of Exercise Science, University of South Carolina, Columbia, SC 29208, USA
| | - Elise A Orloff
- Department of Exercise Science, University of South Carolina, Columbia, SC 29208, USA
| | - Ashley V Haas
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Andrew R Patrick
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Sona Chowdhary
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Barbara C Waldman
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Alan S Waldman
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA.
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A trifunctional Pt(II) complex alleviates the NHEJ/HR-related DSBs repairs to evade cisplatin-resistance in NSCLC. Bioorg Chem 2020; 104:104210. [PMID: 32920356 DOI: 10.1016/j.bioorg.2020.104210] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Revised: 07/29/2020] [Accepted: 08/17/2020] [Indexed: 01/26/2023]
Abstract
Cisplatin, a representative of platinum-based drug, is clinically and widely used in the treatment of various types of malignant cancer. However, its non-selectivity to almost all the cell lines and resistance in long-term use severely limit its scope of use. As biotin-specific uptake systems are overexpressed in many types of tumors but rarely occur in normal tissues, making biotin a promising target for cancer treatment. In the study, we synthesized the Pt(II) complex C2 and determined its biological activities. The existence of biotin enhanced the ability of the complex to target tumors, while the introduction of a naphthalimide compound makes it possible to diagnose tumors and monitor their progress. We have also introduced a known Pt(II) complex DN604, which not only retains the excellent cytotoxicity of platinum drugs, but also inhibits the expression of DNA double-strand breaks (DSBs) repair-related NHEJ protein Ku70 and HR protein Rad51. In summary, we report a novel trifunctional Pt(II) complex that could target tumor cells, monitor tumor progression, and reverse DSBs repair-induced cisplatin-resistance.
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Gao SS, Guan H, Yan S, Hu S, Song M, Guo ZP, Xie DF, Liu Y, Liu X, Zhang S, Zhou PK. TIP60 K430 SUMOylation attenuates its interaction with DNA-PKcs in S-phase cells: Facilitating homologous recombination and emerging target for cancer therapy. SCIENCE ADVANCES 2020; 6:eaba7822. [PMID: 32832608 PMCID: PMC7439314 DOI: 10.1126/sciadv.aba7822] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Accepted: 06/01/2020] [Indexed: 05/12/2023]
Abstract
Nonhomologous end joining (NHEJ) and homologous recombination (HR) are major repair pathways of DNA double-strand breaks (DSBs). The pathway choice of HR and NHEJ is tightly regulated in cellular response to DNA damage. Here, we demonstrate that the interaction of TIP60 with DNA-PKcs is attenuated specifically in S phase, which facilitates HR pathway activation. SUMO2 modification of TIP60 K430 mediated by PISA4 E3 ligase blocks its interaction with DNA-PKcs, whereas TIP60 K430R mutation recovers its interaction with DNA-PKcs, which results in abnormally increased phosphorylation of DNA-PKcs S2056 in S phase and marked inhibition of HR efficiency, but barely affects NHEJ activity. TIP60 K430R mutant cancer cells are more sensitive to radiation and PARP inhibitors in cancer cell killing and tumor growth inhibition. Collectively, coordinated regulation of TIP60 and DNA-PKcs facilitates HR pathway choice in S-phase cells. TIP60 K430R mutant is a potential target of radiation and PARPi cancer therapy.
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Affiliation(s)
- Shan-Shan Gao
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China
| | - Hua Guan
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China
| | - Shuang Yan
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China
- Institute for Environmental Medicine and Radiation Hygiene, School of Public Health, University of South China, Hengyang, Hunan Province 421001, P. R. China
| | - Sai Hu
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China
| | - Man Song
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China
| | - Zong-Pei Guo
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China
| | - Da-Fei Xie
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China
| | - Yike Liu
- Institute for Chemical Carcinogenesis, State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Xinzao, Panyu District, Guangzhou 511436, P. R. China
| | - Xiaodan Liu
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China
| | - Shimeng Zhang
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China
| | - Ping-Kun Zhou
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China
- Institute for Chemical Carcinogenesis, State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Xinzao, Panyu District, Guangzhou 511436, P. R. China
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Tsai L, Lopezcolorado F, Bhargava R, Mendez-Dorantes C, Jahanshir E, Stark J. RNF8 has both KU-dependent and independent roles in chromosomal break repair. Nucleic Acids Res 2020; 48:6032-6052. [PMID: 32427332 PMCID: PMC7293022 DOI: 10.1093/nar/gkaa380] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2019] [Revised: 04/10/2020] [Accepted: 04/30/2020] [Indexed: 12/19/2022] Open
Abstract
Chromosomal double strand breaks (DSBs) can initiate several signaling events, such as ubiquitination, however the precise influence of such signaling on DSB repair outcomes remains poorly understood. With an RNA interference screen, we found that the E3 ubiquitin ligase RNF8 suppresses a deletion rearrangement mediated by canonical non-homologous end joining (C-NHEJ). We also found that RNF8 suppresses EJ without insertion/deletion mutations, which is a hallmark of C-NHEJ. Conversely, RNF8 promotes alternative EJ (ALT-EJ) events involving microhomology that is embedded from the edge of the DSB. These ALT-EJ events likely require limited end resection, whereas RNF8 is not required for single-strand annealing repair involving extensive end resection. Thus, RNF8 appears to specifically facilitate repair events requiring limited end resection, which we find is dependent on the DSB end protection factor KU. However, we also find that RNF8 is important for homology-directed repair (HDR) independently of KU, which appears linked to promoting PALB2 function. Finally, the influence of RNF8 on EJ is distinct from 53BP1 and the ALT-EJ factor, POLQ. We suggest that RNF8 mediates both ALT-EJ and HDR, but via distinct mechanisms, since only the former is dependent on KU.
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Affiliation(s)
- Linda Jillianne Tsai
- Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
- Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
| | | | - Ragini Bhargava
- Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
- Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
| | - Carlos Mendez-Dorantes
- Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
- Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
| | - Eva Jahanshir
- Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
| | - Jeremy M Stark
- Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
- Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
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Lin A, Sheltzer JM. Discovering and validating cancer genetic dependencies: approaches and pitfalls. Nat Rev Genet 2020; 21:671-682. [DOI: 10.1038/s41576-020-0247-7] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/07/2020] [Indexed: 12/21/2022]
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Dodsworth BT, Hatje K, Meyer CA, Flynn R, Cowley SA. Rates of homology directed repair of CRISPR-Cas9 induced double strand breaks are lower in naïve compared to primed human pluripotent stem cells. Stem Cell Res 2020; 46:101852. [PMID: 32521498 PMCID: PMC7347009 DOI: 10.1016/j.scr.2020.101852] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/29/2019] [Revised: 02/14/2020] [Accepted: 05/20/2020] [Indexed: 11/21/2022] Open
Abstract
Kinetics of Cas9-induced double strand break repair in conventional hPSC. Homology directed repair to resolve Cas9-induced double strand breaks is 40% lower in naïve hPSC compared to conventional hPSC. Naïve hPSC (4iLA) have a higher number of cells in G1 phase of the cell cycle. Gene editing in human pluripotent stem cells (hPSC) is a powerful tool for understanding biology, for drug discovery and gene therapy. Naïve hPSC have been suggested to be superior for gene editing compared to conventional ‘primed’ hPSC. Using droplet digital PCR, we uncover the kinetics of Cas9-induced double strand break repair in conventional hPSC. Cut but unrepaired alleles reach their maximum after 12–24 h. Homology directed repair plateaus after 24 h, whereas repair by non-homologous end joining continues until 48 h after Cas9 introduction. Using this method, we demonstrate that the rate of homology directed repair to resolve Cas9-induced double strand breaks is 40% lower in naïve hPSC compared to conventional hPSC, correlating with, and feasibly explained by, a higher number of cells in G1 phase of the cell cycle in naïve hPSC. Therefore, naïve hPSC are less efficient for CRISPR/Cas9-mediated homology directed repair.
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Affiliation(s)
- Benjamin T Dodsworth
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE, United Kingdom; Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland
| | - Klas Hatje
- Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland
| | - Claas Aiko Meyer
- Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland
| | - Rowan Flynn
- Censo Biotechnologies, Roslin Innovation Centre Charnock Bradley Building, Easter Bush Campus EH25 9RG, United Kingdom
| | - Sally A Cowley
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE, United Kingdom.
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Zhu X, Cong J, Lin Z, Sun J, Yang B, Li A. Inhibition of HMGB1 Overcomes Resistance to Radiation and Chemotherapy in Nasopharyngeal Carcinoma. Onco Targets Ther 2020; 13:4189-4199. [PMID: 32523355 PMCID: PMC7236242 DOI: 10.2147/ott.s239243] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2019] [Accepted: 03/25/2020] [Indexed: 01/25/2023] Open
Abstract
Objective This study aimed to investigate the effect of high mobility group protein B1 (HMGB1) on chemoresistance and radioresistance in nasopharyngeal carcinoma (NPC). Materials and Methods HMGB1-knockout HK1 cell lines were generated using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. Western blotting was used to evaluate the protein expression level of HMGB1. DNA repair efficiency of non-homologous end joining (NHEJ) and homologous recombination (HR) was monitored through NHEJ and HR reporter assay. Cellular protein–protein interaction between HMGB1 and NHEJ apparatus was determined by immunoprecipitation. Direct protein–protein interaction was examined by affinity capture assay with purified protein. Protein-DNA binding was evaluated by chromatin fractionation assay. Cell viability assay was employed to measure cell sensitivity to ionizing radiation (IR) or cisplatin. Results HMGB1-knockout NPC cells showed significant decrease in NHEJ efficiency. HMGB1 immunoprecipitated NHEJ key factors in NPC cells and promoted DNA-binding activity of Ku70. Mutational analysis revealed that serine 155 of Ku70 was required for its direct interaction with HMGB1. HMGB1 was highly expressed in radio- and chemoresistant NPC cells. Deficiency of HMGB1 sensitized wild-type (WT) and resistant NPC cells to IR and cisplatin. Glycyrrhizin, which is HMGB1 inhibitor, impaired DNA binding of HMGB1 and exhibited excellent synergy with IR and cisplatin. Conclusion HMGB1 promotes NHEJ via interaction with Ku70 resulting in resistance to IR and cisplatin. Inhibition of HMGB1 by glycyrrhizin is a potential therapeutic regimen to treat cisplatin and IR resistant NPC patients.
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Affiliation(s)
- Xuewei Zhu
- Department of Otolaryngology Head and Neck Surgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin, People's Republic of China
| | - Jianan Cong
- Department of Ophthalmology, Changchun City Central Hospital, Changchun, Jilin, People's Republic of China
| | - Zhang Lin
- Department of Ophthalmology, China-Japan Union Hospital of Jilin University, Changchun, Jilin, People's Republic of China
| | - Jing Sun
- Department of Biochemistry and Molecular Biology, The George Washington University, Washington, DC, USA
| | - Ben Yang
- Department of Ophthalmology, China-Japan Union Hospital of Jilin University, Changchun, Jilin, People's Republic of China
| | - Aipeng Li
- Department of Ophthalmology, The First Hospital of Jilin University, Changchun, Jilin, People's Republic of China
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Park YJ, Kim TS, Kim EH, Kim HD, Kim J. Ribosomal protein S3 is a novel negative regulator of non-homologous end joining repair of DNA double-strand breaks. FASEB J 2020; 34:8102-8113. [PMID: 32297663 DOI: 10.1096/fj.201903245r] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2019] [Revised: 03/16/2020] [Accepted: 03/31/2020] [Indexed: 11/11/2022]
Abstract
DNA double-strand breaks (DSBs) are one of the most serious types of DNA damage. However, multiple repair pathways are present in cells to ensure rapid and appropriate repair of DSBs. Pathway selection depends on several factors including cell type, cell cycle phase, and damage severity. Ribosomal protein S3 (rpS3), a component of the 40S small ribosomal subunit, is a multi-functional protein primarily involved in protein synthesis. rpS3 is also involved in the mediation of various extra-ribosomal pathways, including DNA damage processing and the stress response. Here, we report that rpS3 is a novel negative regulator of non-homologous end joining (NHEJ)-mediated repair of DSBs. We found that rpS3 interacts with the Ku heterodimers of the DNA-dependent protein kinase (DNA-PK) complex and slows down NHEJ ligation reactions, ultimately triggering p53-dependent cell death following treatment with high-dose ionizing radiation. After DSB formation, DNA-PK phosphorylates rpS3, which consequently reduces the binding of rpS3 to the Ku complex. We hypothesized that rpS3 may play a role in DSB repair by repressing NHEJ, while inducing other repair pathways, and by initiating DSB-induced cell death in response to severe DNA damage.
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Affiliation(s)
- Yong Jun Park
- Laboratory of Biochemistry, Division of Life Sciences, Korea University, Seoul, Republic of Korea
| | - Tae-Sung Kim
- Laboratory of Biochemistry, Division of Life Sciences, Korea University, Seoul, Republic of Korea
| | - Eun-Ho Kim
- Laboratory of Biochemistry, Division of Life Sciences, Korea University, Seoul, Republic of Korea
| | | | - Joon Kim
- Laboratory of Biochemistry, Division of Life Sciences, Korea University, Seoul, Republic of Korea.,HAEL Lab, Korea University, Seoul, Korea
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Deng K, Feng W, Liu X, Su X, Zuo E, Du S, Huang Y, Shi D, Lu F. Anti-silencing factor 1A is associated with genome stability maintenance of mouse preimplantation embryos†. Biol Reprod 2020; 102:817-827. [PMID: 31916576 DOI: 10.1093/biolre/ioaa001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2019] [Revised: 10/07/2019] [Accepted: 01/03/2020] [Indexed: 12/26/2022] Open
Abstract
Genome stability is critical for the normal development of preimplantation embryos, as DNA damages may result in mutation and even embryo lethality. Anti-silencing factor 1A (ASF1A) is a histone chaperone and enriched in the MII oocytes as a maternal factor, which may be associated with the maintenance of genome stability. Thus, this study was undertaken to explore the role of ASF1A in maintaining the genome stability of early mouse embryos. The ASF1A expressed in the preimplantation embryos and displayed a dynamic pattern throughout the early embryonic development. Inhibition of ASF1A expression decreased embryonic development and increased DNA damages. Overexpression of ASF1A improved the developmental potential and decreased DNA damages. When 293T cells that had been integrated with RGS-NHEJ were co-transfected with plasmids of pcDNA3.1-ASF1A, gRNA-NHEJ, and hCas9, less cells expressed eGFP, indicating that non-homologous end joining was reduced by ASF1A. When 293T cells were co-transfected with plasmids of HR-donor, gRNA-HR, hCas9, and pcDNA3.1-ASF1A, more cells expressed eGFP, indicating that homologous recombination (HR) was enhanced by ASF1A. These results indicate that ASF1A may be associated with the genome stability maintenance of early mouse embryos and this action may be mediated by promoting DNA damage repair through HR pathway.
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Affiliation(s)
- Kai Deng
- Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China and
| | - Wanyou Feng
- Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China and
| | - Xiaohua Liu
- Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China and
| | - Xiaoping Su
- Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China and
| | - Erwei Zuo
- Center for Animal Genomics, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China
| | - Shanshan Du
- Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China and
| | - Yongjun Huang
- Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China and
| | - Deshun Shi
- Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China and
| | - Fenghua Lu
- Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China and
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Patel A, Seraia E, Ebner D, Ryan AJ. Adefovir dipivoxil induces DNA replication stress and augments ATR inhibitor-related cytotoxicity. Int J Cancer 2020; 147:1474-1484. [PMID: 32159854 DOI: 10.1002/ijc.32966] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2019] [Revised: 02/20/2020] [Accepted: 03/02/2020] [Indexed: 12/20/2022]
Abstract
Replication stress is a common feature of cancer cells. Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signalling, a DNA damage repair (DDR) pathway, is activated by regions of single-stranded DNA (ssDNA) that can arise during replication stress. ATR delays cell cycle progression and prevents DNA replication fork collapse, which prohibits cell death and promotes proliferation. Several ATR inhibitors have been developed in order to restrain this protective mechanism in tumours. It is known, however, that despite other effective anticancer chemotherapy treatments targeting DDR pathways, resistance occurs. This begets the need to identify combination treatments to overcome resistance and prevent tumour cell growth. We conducted a drug screen to identify potential synergistic combination treatments by screening an ATR inhibitor (VE822) together with compounds from a bioactive small molecule library. The screen identified adefovir dipivoxil, a reverse transcriptase inhibitor and nucleoside analogue, as a compound that has increased cytotoxicity in the presence of ATR, but not ATM or DNA-dependant protein kinase (DNA-PK) inhibition. Here we demonstrate that adefovir dipivoxil induces DNA replication stress, activates ATR signalling and stalls cells in S phase. This simultaneous induction of replication stress and inhibition of ATR signalling lead to a marked increase in pan-nuclear γH2AX-positive cells, ssDNA accumulation and cell death, indicative of replication catastrophe.
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Affiliation(s)
- Agata Patel
- The Department of Oncology, Oxford Institute for Radiation Oncology, University of Oxford, Oxford, United Kingdom
| | - Elena Seraia
- The Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, United Kingdom
| | - Daniel Ebner
- The Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, United Kingdom
| | - Anderson Joseph Ryan
- The Department of Oncology, Oxford Institute for Radiation Oncology, University of Oxford, Oxford, United Kingdom
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Furukawa S, Nagamatsu A, Nenoi M, Fujimori A, Kakinuma S, Katsube T, Wang B, Tsuruoka C, Shirai T, Nakamura AJ, Sakaue-Sawano A, Miyawaki A, Harada H, Kobayashi M, Kobayashi J, Kunieda T, Funayama T, Suzuki M, Miyamoto T, Hidema J, Yoshida Y, Takahashi A. Space Radiation Biology for "Living in Space". BIOMED RESEARCH INTERNATIONAL 2020; 2020:4703286. [PMID: 32337251 PMCID: PMC7168699 DOI: 10.1155/2020/4703286] [Citation(s) in RCA: 70] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Accepted: 03/13/2020] [Indexed: 12/16/2022]
Abstract
Space travel has advanced significantly over the last six decades with astronauts spending up to 6 months at the International Space Station. Nonetheless, the living environment while in outer space is extremely challenging to astronauts. In particular, exposure to space radiation represents a serious potential long-term threat to the health of astronauts because the amount of radiation exposure accumulates during their time in space. Therefore, health risks associated with exposure to space radiation are an important topic in space travel, and characterizing space radiation in detail is essential for improving the safety of space missions. In the first part of this review, we provide an overview of the space radiation environment and briefly present current and future endeavors that monitor different space radiation environments. We then present research evaluating adverse biological effects caused by exposure to various space radiation environments and how these can be reduced. We especially consider the deleterious effects on cellular DNA and how cells activate DNA repair mechanisms. The latest technologies being developed, e.g., a fluorescent ubiquitination-based cell cycle indicator, to measure real-time cell cycle progression and DNA damage caused by exposure to ultraviolet radiation are presented. Progress in examining the combined effects of microgravity and radiation to animals and plants are summarized, and our current understanding of the relationship between psychological stress and radiation is presented. Finally, we provide details about protective agents and the study of organisms that are highly resistant to radiation and how their biological mechanisms may aid developing novel technologies that alleviate biological damage caused by radiation. Future research that furthers our understanding of the effects of space radiation on human health will facilitate risk-mitigating strategies to enable long-term space and planetary exploration.
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Affiliation(s)
- Satoshi Furukawa
- Japan Aerospace Exploration Agency, 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505, Japan
| | - Aiko Nagamatsu
- Japan Aerospace Exploration Agency, 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505, Japan
| | - Mitsuru Nenoi
- National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology (QST), 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
| | - Akira Fujimori
- National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology (QST), 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
| | - Shizuko Kakinuma
- National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology (QST), 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
| | - Takanori Katsube
- National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology (QST), 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
| | - Bing Wang
- National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology (QST), 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
| | - Chizuru Tsuruoka
- National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology (QST), 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
| | - Toshiyuki Shirai
- National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology (QST), 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
| | - Asako J. Nakamura
- Department of Biological Sciences, College of Science, Ibaraki University, 2-1-1, Bunkyo, Mito, Ibaraki 310-8512, Japan
| | - Asako Sakaue-Sawano
- Lab for Cell Function and Dynamics, CBS, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
| | - Atsushi Miyawaki
- Lab for Cell Function and Dynamics, CBS, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
| | - Hiroshi Harada
- Radiation Biology Center, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
| | - Minoru Kobayashi
- Radiation Biology Center, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
| | - Junya Kobayashi
- Radiation Biology Center, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
| | - Takekazu Kunieda
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Tomoo Funayama
- Takasaki Advanced Radiation Research Institute, QST, 1233 Watanuki-machi, Takasaki, Gunma 370-1292, Japan
| | - Michiyo Suzuki
- Takasaki Advanced Radiation Research Institute, QST, 1233 Watanuki-machi, Takasaki, Gunma 370-1292, Japan
| | - Tatsuo Miyamoto
- Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan
| | - Jun Hidema
- Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan
- Division for the Establishment of Frontier Sciences of the Organization for Advanced Studies, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan
| | - Yukari Yoshida
- Gunma University Heavy Ion Medical Center, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
| | - Akihisa Takahashi
- Gunma University Heavy Ion Medical Center, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
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50
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Sun Y, Saha S, Wang W, Saha LK, Huang SYN, Pommier Y. Excision repair of topoisomerase DNA-protein crosslinks (TOP-DPC). DNA Repair (Amst) 2020; 89:102837. [PMID: 32200233 DOI: 10.1016/j.dnarep.2020.102837] [Citation(s) in RCA: 69] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2020] [Revised: 02/22/2020] [Accepted: 02/25/2020] [Indexed: 12/13/2022]
Abstract
Topoisomerases are essential enzymes solving DNA topological problems such as supercoils, knots and catenanes that arise from replication, transcription, chromatin remodeling and other nucleic acid metabolic processes. They are also the targets of widely used anticancer drugs (e.g. topotecan, irinotecan, enhertu, etoposide, doxorubicin, mitoxantrone) and fluoroquinolone antibiotics (e.g. ciprofloxacin and levofloxacin). Topoisomerases manipulate DNA topology by cleaving one DNA strand (TOP1 and TOP3 enzymes) or both in concert (TOP2 enzymes) through the formation of transient enzyme-DNA cleavage complexes (TOPcc) with phosphotyrosyl linkages between DNA ends and the catalytic tyrosyl residue of the enzymes. Failure in the self-resealing of TOPcc results in persistent TOPcc (which we refer it to as topoisomerase DNA-protein crosslinks (TOP-DPC)) that threaten genome integrity and lead to cancers and neurodegenerative diseases. The cell prevents the accumulation of topoisomerase-mediated DNA damage by excising TOP-DPC and ligating the associated breaks using multiple pathways conserved in eukaryotes. Tyrosyl-DNA phosphodiesterases (TDP1 and TDP2) cleave the tyrosyl-DNA bonds whereas structure-specific endonucleases such as Mre11 and XPF (Rad1) incise the DNA phosphodiester backbone to remove the TOP-DPC along with the adjacent DNA segment. The proteasome and metalloproteases of the WSS1/Spartan family typify proteolytic repair pathways that debulk TOP-DPC to make the peptide-DNA bonds accessible to the TDPs and endonucleases. The purpose of this review is to summarize our current understanding of how the cell excises TOP-DPC and why, when and where the cell recruits one specific mechanism for repairing topoisomerase-mediated DNA damage, acquiring resistance to therapeutic topoisomerase inhibitors and avoiding genomic instability, cancers and neurodegenerative diseases.
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Affiliation(s)
- Yilun Sun
- Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States
| | - Sourav Saha
- Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States
| | - Wenjie Wang
- Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States
| | - Liton Kumar Saha
- Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States
| | - Shar-Yin Naomi Huang
- Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States
| | - Yves Pommier
- Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States.
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