|
1
|
Sun F, Li W, Du R, Liu M, Cheng Y, Ma J, Yan S. Impact of glycolysis enzymes and metabolites in regulating DNA damage repair in tumorigenesis and therapy. Cell Commun Signal 2025; 23:44. [PMID: 39849559 PMCID: PMC11760674 DOI: 10.1186/s12964-025-02047-9] [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: 12/07/2024] [Accepted: 01/16/2025] [Indexed: 01/25/2025] Open
Abstract
Initially, it was believed that glycolysis and DNA damage repair (DDR) were two distinct biological processes that independently regulate tumor progression. The former metabolic reprogramming rapidly generates energy and generous intermediate metabolites, supporting the synthetic metabolism and proliferation of tumor cells. While the DDR plays a pivotal role in preserving genomic stability, thus resisting cellular senescence and cell death under both physiological and radio-chemotherapy conditions. Recently, an increasing number of studies have shown closely correlation between these two biological processes, and then promoting tumor progression. For instance, lactic acid, the product of glycolysis, maintains an acidic tumor microenvironment that not only fosters cell proliferation and invasion but also facilitates DDR by enhancing AKT activity. Here, we provide a comprehensive overview of the enzymes and metabolites involved in glycolysis, along with the primary methods for DDR. Meanwhile, this review explores existing knowledge of glycolysis enzymes and metabolites in regulating DDR. Moreover, considering the significant roles of glycolysis and DDR in tumor development and radio-chemotherapy resistance, the present review discusses effective direct or indirect therapeutic strategies targeted to glycolysis and DDR.
Collapse
Affiliation(s)
- Fengyao Sun
- Precision Medicine Laboratory for Chronic Non-Communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Wen Li
- Precision Medicine Laboratory for Chronic Non-Communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Ruihang Du
- Precision Medicine Laboratory for Chronic Non-Communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Mingchan Liu
- Precision Medicine Laboratory for Chronic Non-Communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Yi Cheng
- Precision Medicine Laboratory for Chronic Non-Communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Jianxing Ma
- Precision Medicine Laboratory for Chronic Non-Communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, 272067, China
| | - Siyuan Yan
- Precision Medicine Laboratory for Chronic Non-Communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, 272067, China.
| |
Collapse
|
|
2
|
Dardare J, Witz A, Betz M, François A, Lamy L, Husson M, Demange J, Rouyer M, Lambert A, Merlin JL, Gilson P, Harlé A. DDB2 expression lights the way for precision radiotherapy response in PDAC cells, with or without olaparib. Cell Death Discov 2024; 10:411. [PMID: 39333096 PMCID: PMC11436999 DOI: 10.1038/s41420-024-02188-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2023] [Revised: 09/16/2024] [Accepted: 09/20/2024] [Indexed: 09/29/2024] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Therapeutic options for PDAC are primarily restricted to surgery in the early stages of the disease or chemotherapy in advanced disease. Only a subset of patients with germline defects in BRCA1/2 genes can potentially benefit from personalized therapy, with the PARP inhibitor olaparib serving as a maintenance treatment for metastatic disease. Although the role of radiotherapy in PDAC remains controversial, the use of radiosensitizers offers hope for improving cancer management. Previously, we have shown that damage-specific DNA binding protein 2 (DDB2) is a potential prognostic and predictive biomarker for chemotherapy response in PDAC. In this study, we investigated the function of DDB2 in radiotherapy response, with and without radiosensitization by olaparib in PDAC cells. Our findings demonstrated DDB2 resistance to radiation effects, thereby improving cell survival and enhancing the repair of ionizing radiation-induced DNA double-strand breaks. We observed that DDB2 expression enhances the cell cycle arrest in the G2 phase by phosphorylating Chk1 and Chk2 cell cycle checkpoints. Additionally, we identified a novel link between DDB2 and PARP1 in the context of radiotherapy, which enhances the expression and activity of PARP1. Our findings highlight the potential of low-DDB2 expression to potentiate the radiosensitization effect of olaparib in PDAC cells. Collectively, this study provides novel insights into the impacts of DDB2 in the radiotherapy response in PDAC, enabling its employment as a potential biomarker to predict resistance to radiation. Furthermore, DDB2 represents a significant step forward in precision radiotherapy by widening the scope of patients who can be benefiting from olaparib as a radiosensitizer. Hence, this research has the potential to enrich the limited use of radiotherapy in the care of patients with PDAC.
Collapse
Affiliation(s)
- Julie Dardare
- Université de Lorraine, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 7039 Centre de Recherche en Automatique de Nancy (CRAN), Nancy, France.
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France.
| | - Andréa Witz
- Université de Lorraine, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 7039 Centre de Recherche en Automatique de Nancy (CRAN), Nancy, France
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| | - Margaux Betz
- Université de Lorraine, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 7039 Centre de Recherche en Automatique de Nancy (CRAN), Nancy, France
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| | - Aurélie François
- Université de Lorraine, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 7039 Centre de Recherche en Automatique de Nancy (CRAN), Nancy, France
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| | - Laureline Lamy
- Université de Lorraine, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 7039 Centre de Recherche en Automatique de Nancy (CRAN), Nancy, France
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| | - Marie Husson
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| | - Jessica Demange
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| | - Marie Rouyer
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| | - Aurélien Lambert
- Département d'oncologie médicale, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| | - Jean-Louis Merlin
- Université de Lorraine, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 7039 Centre de Recherche en Automatique de Nancy (CRAN), Nancy, France
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| | - Pauline Gilson
- Université de Lorraine, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 7039 Centre de Recherche en Automatique de Nancy (CRAN), Nancy, France
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| | - Alexandre Harlé
- Université de Lorraine, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 7039 Centre de Recherche en Automatique de Nancy (CRAN), Nancy, France
- Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandœuvre-lès-Nancy, France
| |
Collapse
|
|
3
|
Shishido K, Reinders A, Asuthkar S. Epigenetic regulation of radioresistance: insights from preclinical and clinical studies. Expert Opin Investig Drugs 2022; 31:1359-1375. [PMID: 36524403 DOI: 10.1080/13543784.2022.2158810] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
INTRODUCTION Oftentimes, radiation therapy (RT) is ineffective due to the development of radioresistance (RR). However, studies have shown that targeting epigenetic modifiers to enhance radiosensitivity represents a promising direction of clinical investigation. AREAS COVERED This review discusses the mechanisms by which epigenetic modifiers alter radiosensitivity through dysregulation of MAPK-ERK and AKT-mTOR signaling. Finally, we discuss the clinical directions for targeting epigenetic modifiers and current radiology techniques used in the clinic. METHODOLOGY We searched PubMed and ScienceDirect databases from April 4th, 2022 to October 18th, 2022. We examined 226 papers related to radioresistance, epigenetics, MAPK, and PI3K/AKT/mTOR signaling. 194 papers were selected for this review. Keywords used for this search include, 'radioresistance,' 'radiosensitivity,' 'radiation,' 'radiotherapy,' 'particle radiation,' 'photon radiation,' 'epigenetic modifiers,' 'MAPK,' 'AKT,' 'mTOR,' 'cancer,' and 'PI3K.' We examined 41 papers related to clinical trials on the aforementioned topics. Outcomes of interest were safety, overall survival (OS), dose-limiting toxicities (DLT), progression-free survival (PFS), and maximum tolerated dose (MTD). EXPERT OPINION Current studies focusing on epigenetic mechanisms of RR strongly support the use of targeting epigenetic modifiers as adjuvants to standard cancer therapies. To further the success of such treatments and their clinical benefit , both preclinical and clinical studies are needed to broaden the scope of known radioresistant mechanisms.
Collapse
Affiliation(s)
- Katherine Shishido
- Department of Cancer Biology and Pharmacology and Department of Pediatrics, University of Illinois College of Medicine Peoria, Peoria, IL, United States of America
| | - Alexis Reinders
- Department of Cancer Biology and Pharmacology and Department of Pediatrics, University of Illinois College of Medicine Peoria, Peoria, IL, United States of America
| | - Swapna Asuthkar
- Department of Cancer Biology and Pharmacology and Department of Pediatrics, University of Illinois College of Medicine Peoria, Peoria, IL, United States of America
| |
Collapse
|
|
4
|
Tamanoi F, Yoshikawa K. Summary of volume 51: DNA damage and double-strand breaks. Enzymes 2022; 52:1-10. [PMID: 36423960 DOI: 10.1016/bs.enz.2022.10.001] [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] [Indexed: 06/16/2023]
Abstract
DNA damage and breaks are events that happen to DNA which exert a variety of influence on cell physiology including inhibition of DNA synthesis, repair response, cell cycle effect and cell death. Thus, it is important to deepened understanding of these events. In volume 51, we discussed topics including (1) assays to detect double-strand breaks, (2) conditions leading to double-strand breaks, (3) effects of irradiation, (4) DNA structure and chromatins, and (5) direct and indirect effect on DNA. Contributing authors and a table of contents for volume 51 are mentioned. We also discuss further issues and topics that need to be featured in future volumes. These include DNA damage sensors, DNA damage response proteins, and double-strand break repair pathways.
Collapse
Affiliation(s)
- Fuyuhiko Tamanoi
- Institute for Integrated Cell-Material Sciences, Institute for Advanced Study, Kyoto University, Kyoto, Japan.
| | - Kenichi Yoshikawa
- Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan; Center for Integrative Medicine and Physics, Kyoto University, Kyoto, Japan
| |
Collapse
|
|
5
|
Waldron E, Tanhehco YC. Under the Hood: The Molecular Biology Driving Gene Therapy for the Treatment of Sickle Cell Disease. Transfus Apher Sci 2022; 61:103566. [DOI: 10.1016/j.transci.2022.103566] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
|
|
6
|
Abstract
Redondoviridae is a newly established family of circular Rep-encoding single-stranded (CRESS) DNA viruses found in the human ororespiratory tract. Redondoviruses were previously found in ∼15% of respiratory specimens from U.S. urban subjects; levels were elevated in individuals with periodontitis or critical illness. Here, we report higher redondovirus prevalence in saliva samples: four rural African populations showed 61 to 82% prevalence, and an urban U.S. population showed 32% prevalence. Longitudinal, limiting-dilution single-genome sequencing revealed diverse strains of both redondovirus species (Brisavirus and Vientovirus) in single individuals, persistence over time, and evidence of intergenomic recombination. Computational analysis of viral genomes identified a recombination hot spot associated with a conserved potential DNA stem-loop structure. To assess the possible role of this site in recombination, we carried out in vitro studies which showed that this potential stem-loop was cleaved by the virus-encoded Rep protein. In addition, in reconstructed reactions, a Rep-DNA covalent intermediate was shown to mediate DNA strand transfer at this site. Thus, redondoviruses are highly prevalent in humans, found in individuals on multiple continents, heterogeneous even within individuals and encode a Rep protein implicated in facilitating recombination. IMPORTANCE Redondoviridae is a recently established family of DNA viruses predominantly found in the human respiratory tract and associated with multiple clinical conditions. In this study, we found high redondovirus prevalence in saliva from urban North American individuals and nonindustrialized African populations in Botswana, Cameroon, Ethiopia, and Tanzania. Individuals on both continents harbored both known redondovirus species. Global prevalence of both species suggests that redondoviruses have long been associated with humans but have remained undetected until recently due to their divergent genomes. By sequencing single redondovirus genomes in longitudinally sampled humans, we found that redondoviruses persisted over time within subjects and likely evolve by recombination. The Rep protein encoded by redondoviruses catalyzes multiple reactions in vitro, consistent with a role in mediating DNA replication and recombination. In summary, we identify high redondovirus prevalence in humans across multiple continents, longitudinal heterogeneity and persistence, and potential mechanisms of redondovirus evolution by recombination.
Collapse
|
|
7
|
Sun B, Chen H, Gao X. Versatile modification of the CRISPR/Cas9 ribonucleoprotein system to facilitate in vivo application. J Control Release 2021; 337:698-717. [PMID: 34364918 DOI: 10.1016/j.jconrel.2021.08.007] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Revised: 08/03/2021] [Accepted: 08/03/2021] [Indexed: 12/26/2022]
Abstract
The development of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems has created a tremendous wave that is sweeping the world of genome editing. The ribonucleoprotein (RNP) method has evolved to be the most advantageous form for in vivo application. Modification of the CRISPR/Cas9 RNP method to adapt delivery through a variety of carriers can either directly improve the stability and specificity of the gene-editing tool in vivo or indirectly endow the system with high gene-editing efficiency that induces few off-target mutations through different delivery methods. The exploration of in vivo applications mediated by various delivery methods lays the foundation for genome research and variety improvements, which is especially promising for better in vivo research in the field of translational biomedicine. In this review, we illustrate the modifiable structures of the Cas9 nuclease and single guide RNA (sgRNA), summarize the latest research progress and discuss the feasibility and advantages of various methods. The highlighted results will enhance our knowledge, stimulate extensive research and application of Cas9 and provide alternatives for the development of rational delivery carriers in multiple fields.
Collapse
Affiliation(s)
- Bixi Sun
- Department of Biopharmacy, School of Pharmaceutical Sciences, Jilin University, 1266 Fujin Road, Changchun 130021, China
| | - Hening Chen
- Department of Biopharmacy, School of Pharmaceutical Sciences, Jilin University, 1266 Fujin Road, Changchun 130021, China
| | - Xiaoshu Gao
- Department of Biopharmacy, School of Pharmaceutical Sciences, Jilin University, 1266 Fujin Road, Changchun 130021, China.
| |
Collapse
|
|
8
|
Luo EWC, Liao ML, Chien CL. Neural differentiation of glioblastoma cell lines via a herpes simplex virus thymidine kinase/ganciclovir system driven by a glial fibrillary acidic protein promoter. PLoS One 2021; 16:e0253008. [PMID: 34370752 PMCID: PMC8351974 DOI: 10.1371/journal.pone.0253008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2021] [Accepted: 05/27/2021] [Indexed: 11/18/2022] Open
Abstract
Glioblastoma is a malignant brain tumor with poor prognosis that rapidly acquires resistance to available clinical treatments. The herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system produces the selective elimination of HSVtk-positive cells and is a candidate for preclinical testing against glioblastoma via its ability to regulate proliferation and differentiation. Therefore, in this study, we aimed to establish a plasmid encoding the HSVtk/GCV system driven by a glial fibrillary acidic protein (GFAP) promoter and verify its possibility of neural differentiation of glioblastoma cell line under the GCV challenge. Four stable clones-N2A-pCMV-HSVtk, N2A-pGFAP-HSVtk, U251-pCMV-HSVtk, and U251-pGFAP-HSVtk-were established from neuronal N2A and glioblastoma U251 cell lines. In vitro GCV sensitivity was assessed by MTT assay for monitoring time- and dosage-dependent cytotoxicity. The capability for neural differentiation in stable glioblastoma clones during GCV treatment was assessed by performing immunocytochemistry for nestin, GFAP, and βIII-tubulin. Under GFAP promoter control, the U251 stable clone exhibited GCV sensitivity, while the neuronal N2A clones were nonreactive. During GCV treatment, cells underwent apoptosis on day 3 and dying cells were identified after day 5. Nestin was increasingly expressed in surviving cells, indicating that the population of neural stem-like cells was enriched. Lower levels of GFAP expression were detected in surviving cells. Furthermore, βIII-tubulin-positive neuron-like cells were identified after GCV treatment. This study established pGFAP-HSVtk-P2A-EGFP plasmids that successfully ablated GFAP-positive glioblastoma cells, but left neuronal N2A cells intact. These data suggest that the neural differentiation of glioblastoma cells can be promoted by treatment with the HSVtk/GCV system.
Collapse
Affiliation(s)
- Elizabeth Wei-Chia Luo
- Graduate Institute of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
- Department of Bioengineering, University of California, Los Angeles, California, United States of America
| | - Meng-Lin Liao
- Graduate Institute of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
- School of Medicine, College of Medicine, I‐Shou University, Kaohsiung, Taiwan
- * E-mail: (CLC); (MLL)
| | - Chung-Liang Chien
- Graduate Institute of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
- * E-mail: (CLC); (MLL)
| |
Collapse
|
|
9
|
Can neural signals override cellular decisions in the presence of DNA damage? DNA Repair (Amst) 2021; 103:103127. [PMID: 33990031 DOI: 10.1016/j.dnarep.2021.103127] [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: 01/31/2021] [Revised: 04/15/2021] [Accepted: 04/16/2021] [Indexed: 11/23/2022]
Abstract
Cells within an organism are in constant crosstalk with their surrounding environment. Short and long-range signals influence cellular behavior associated with division, differentiation, and death. This crosstalk among cells underlies tissue renewal to guarantee faithful replacement of old or damaged cells over many years. Renewing tissues also offer recurrent opportunities for DNA damage and cellular transformation that tend to occur with aging. Most cells with extensive DNA damage have limited options such as halting cell cycle to repair DNA, undergo senescence, or programmed cell death. However, in some cases cells carrying toxic forms of DNA damage survive and proliferate. The underlying factors driving survival and proliferation of cells with DNA damage remain unknown. Here we discuss potential roles the nervous system may play in influencing the fate of cells with DNA damage. We present a brief survey highlighting the implications the nervous system has in regeneration, regulation of stem cells, modulation of the immune system, and its contribution to cancer progression. Finally, we propose the use of planarian flatworms as a convenient model organism to molecularly dissect the influence of neural signals over cellular fate regulation in the presence of DNA damage.
Collapse
|
|
10
|
Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing. Theranostics 2021; 11:614-648. [PMID: 33391496 PMCID: PMC7738854 DOI: 10.7150/thno.47007] [Citation(s) in RCA: 216] [Impact Index Per Article: 54.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Accepted: 08/31/2020] [Indexed: 12/26/2022] Open
Abstract
CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed.
Collapse
Affiliation(s)
- Song Zhang
- South China Advanced Institute for Soft Matter Science and Technology, School of Molecular Science and Engineering, South China University of Technology, Guangzhou 510640, China
| | - Jiangtao Shen
- The Second People's Hospital of Taizhou affiliated to Yangzhou University, Taizhou, 225500, China
| | - Dali Li
- Shanghai Key Laboratory of Regulatory Biology, East China Normal University, Shanghai 200241, China
| | - Yiyun Cheng
- South China Advanced Institute for Soft Matter Science and Technology, School of Molecular Science and Engineering, South China University of Technology, Guangzhou 510640, China
- Shanghai Key Laboratory of Regulatory Biology, East China Normal University, Shanghai 200241, China
| |
Collapse
|
|
11
|
Moscoso CG, Steer CJ. Liver targeted gene therapy: Insights into emerging therapies. DRUG DISCOVERY TODAY. TECHNOLOGIES 2020; 34:9-19. [PMID: 33357766 DOI: 10.1016/j.ddtec.2020.11.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/16/2020] [Revised: 10/23/2020] [Accepted: 11/03/2020] [Indexed: 12/24/2022]
Abstract
The large number of monogenic metabolic disorders originating in the liver poses a unique opportunity for development of gene therapy modalities to pursue curative approaches. Various disorders have been successfully treated via liver-directed gene therapy, though most of the advances have been in animal models, with only limited success in clinical trials. Pre-clinical data in animals using non-viral approaches, including the Sleeping Beauty transposon system, are discussed. The various advances with viral vectors for liver-directed gene therapy are also a focus of this review, including retroviral, adenoviral, recombinant adeno-associated viral, and SV40 vectors. Genome editing techniques, including zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats (CRISPR), are also described. Further, the various controversies in the field with regards to somatic vs. germline editing using CRISPR in humans are explored, while also highlighting the myriad of preclinical advances. Lastly, newer technologies are reviewed, including base editing and prime editing, which use CRISPR with exciting adjunctive properties to avoid double-stranded breaks and thus the recruitment of endogenous repair mechanisms. While encouraging results have been achieved recently, there are still significant challenges to overcome prior to the broad use of vector-based and genome editing techniques in the clinical arena. As these technologies mature, the promise of a cure for many disabling inherited metabolic disorders is within reach, and urgently needed.
Collapse
Affiliation(s)
- Carlos G Moscoso
- Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
| | - Clifford J Steer
- Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA; Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455 USA.
| |
Collapse
|
|
12
|
Huang RX, Zhou PK. DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer. Signal Transduct Target Ther 2020; 5:60. [PMID: 32355263 PMCID: PMC7192953 DOI: 10.1038/s41392-020-0150-x] [Citation(s) in RCA: 613] [Impact Index Per Article: 122.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2020] [Revised: 02/20/2020] [Accepted: 03/16/2020] [Indexed: 12/19/2022] Open
Abstract
Radiotherapy is one of the most common countermeasures for treating a wide range of tumors. However, the radioresistance of cancer cells is still a major limitation for radiotherapy applications. Efforts are continuously ongoing to explore sensitizing targets and develop radiosensitizers for improving the outcomes of radiotherapy. DNA double-strand breaks are the most lethal lesions induced by ionizing radiation and can trigger a series of cellular DNA damage responses (DDRs), including those helping cells recover from radiation injuries, such as the activation of DNA damage sensing and early transduction pathways, cell cycle arrest, and DNA repair. Obviously, these protective DDRs confer tumor radioresistance. Targeting DDR signaling pathways has become an attractive strategy for overcoming tumor radioresistance, and some important advances and breakthroughs have already been achieved in recent years. On the basis of comprehensively reviewing the DDR signal pathways, we provide an update on the novel and promising druggable targets emerging from DDR pathways that can be exploited for radiosensitization. We further discuss recent advances identified from preclinical studies, current clinical trials, and clinical application of chemical inhibitors targeting key DDR proteins, including DNA-PKcs (DNA-dependent protein kinase, catalytic subunit), ATM/ATR (ataxia-telangiectasia mutated and Rad3-related), the MRN (MRE11-RAD50-NBS1) complex, the PARP (poly[ADP-ribose] polymerase) family, MDC1, Wee1, LIG4 (ligase IV), CDK1, BRCA1 (BRCA1 C terminal), CHK1, and HIF-1 (hypoxia-inducible factor-1). Challenges for ionizing radiation-induced signal transduction and targeted therapy are also discussed based on recent achievements in the biological field of radiotherapy.
Collapse
Affiliation(s)
- Rui-Xue Huang
- Department of Occupational and Environmental Health, Xiangya School of Public Health, Central South University, 410078, Changsha, People's Republic of China
| | - Ping-Kun Zhou
- Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, AMMS, 100850, Beijing, People's Republic of China.
- Institute for Chemical Carcinogenesis, State Key Laboratory of Respiratory, Guangzhou Medical University, 511436, Guangzhou, People's Republic of China.
| |
Collapse
|
|
13
|
Ewart D, Peterson EJ, Steer CJ. A new era of genetic engineering for autoimmune and inflammatory diseases. Semin Arthritis Rheum 2019; 49:e1-e7. [DOI: 10.1016/j.semarthrit.2019.05.004] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2019] [Accepted: 05/09/2019] [Indexed: 02/06/2023]
|
|
14
|
Ewart DT, Peterson EJ, Steer CJ. Gene editing for inflammatory disorders. Ann Rheum Dis 2019; 78:6-15. [PMID: 30077989 DOI: 10.1136/annrheumdis-2018-213454] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2018] [Revised: 07/02/2018] [Accepted: 07/03/2018] [Indexed: 12/24/2022]
Abstract
Technology for precise and efficient genetic editing is constantly evolving and is now capable of human clinical applications. Autoimmune and inflammatory diseases are chronic, disabling, sometimes life-threatening, conditions that feature heritable components. Both primary genetic lesions and the inflammatory pathobiology underlying these diseases represent fertile soil for new therapies based on the capabilities of gene editing. The ability to orchestrate precise targeted modifications to the genome will likely enable cell-based therapies for inflammatory diseases such as monogenic autoinflammatory disease, acquired autoimmune disease and for regenerative medicine in the setting of an inflammatory environment. Here, we discuss recent advances in genome editing and their evolving applications in immunoinflammatory diseases. Strengths and limitations of older genetic modification tools are compared with CRISPR/Cas9, base editing, RNA editing, targeted activators and repressors of transcription and targeted epigenetic modifiers. Commonly employed delivery vehicles to target cells or tissues of interest with genetic modification machinery, including viral, non-viral and cellular vectors, are described. Finally, applications in animal and human models of inflammatory diseases are discussed. Use of chimeric autoantigen receptor T cells, correction of monogenic diseases with genetically edited haematopoietic stem and progenitor cells, engineering of induced pluripotent stem cells and ex vivo expansion and modification of regulatory T cells for a range of chronic inflammatory diseases are reviewed.
Collapse
Affiliation(s)
- David T Ewart
- Division of Rheumatic and Autoimmune Diseases, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA
| | - Erik J Peterson
- Division of Rheumatic and Autoimmune Diseases, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA
| | - Clifford J Steer
- Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA
- Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota, USA
| |
Collapse
|
|
15
|
Aravalli RN, Steer CJ. CRISPR/Cas9 therapeutics for liver diseases. J Cell Biochem 2018; 119:4265-4278. [PMID: 29266637 DOI: 10.1002/jcb.26627] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2017] [Accepted: 12/18/2017] [Indexed: 12/20/2022]
Abstract
The development of innovative genome editing techniques in recent years has revolutionized the field of biomedicine. Among the novel approaches, the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas9) technology has become the most popular, in part due to its matchless ability to carry out gene editing at the target site with great precision. With considerable successes in animal and preclinical studies, CRISPR/Cas9-mediated gene editing has paved the way for its use in human trials, including patients with a variety of liver diseases. Gene editing is a logical therapeutic approach for liver diseases because many metabolic and acquired disorders are caused by mutations within a single gene. In this review, we provide an overview on current and emerging therapeutic strategies for the treatment of liver diseases using the CRISPR/Cas9 technology.
Collapse
Affiliation(s)
- Rajagopal N Aravalli
- Department of Electrical and Computer Engineering, University of Minnesota, Minneapolis, Minnesota
| | - Clifford J Steer
- Department of Medicine, University of Minnesota, Minneapolis, Minnesota.,Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota
| |
Collapse
|
|
16
|
Baird DM, Hendrickson EA. Telomeres and Chromosomal Translocations : There's a Ligase at the End of the Translocation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2018; 1044:89-112. [PMID: 29956293 DOI: 10.1007/978-981-13-0593-1_7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Chromosomal translocations are now well understood to not only constitute signature molecular markers for certain human cancers but often also to be causative in the genesis of that tumor. Despite the obvious importance of such events, the molecular mechanism of chromosomal translocations in human cells remains poorly understood. Part of the explanation for this dearth of knowledge is due to the complexity of the reaction and the need to archaeologically work backwards from the final product (a translocation) to the original unrearranged chromosomes to infer mechanism. Although not definitive, these studies have indicated that the aberrant usage of endogenous DNA repair pathways likely lies at the heart of the problem. An equally obfuscating aspect of this field, however, has also originated from the unfortunate species-specific differences that appear to exist in the relevant model systems that have been utilized to investigate this process. Specifically, yeast and murine systems (which are often used by basic science investigators) rely on different DNA repair pathways to promote chromosomal translocations than human somatic cells. In this chapter, we will review some of the basic concepts of chromosomal translocations and the DNA repair systems thought to be responsible for their genesis with an emphasis on underscoring the differences between other species and human cells. In addition, we will focus on a specific subset of translocations that involve the very end of a chromosome (a telomere). A better understanding of the relationship between DNA repair pathways and chromosomal translocations is guaranteed to lead to improved therapeutic treatments for cancer.
Collapse
Affiliation(s)
- Duncan M Baird
- Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, UK
| | - Eric A Hendrickson
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN, USA.
| |
Collapse
|
|
17
|
Saito S, Maeda R, Adachi N. Dual loss of human POLQ and LIG4 abolishes random integration. Nat Commun 2017; 8:16112. [PMID: 28695890 PMCID: PMC5508229 DOI: 10.1038/ncomms16112] [Citation(s) in RCA: 71] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2017] [Accepted: 05/30/2017] [Indexed: 12/16/2022] Open
Abstract
Homologous recombination-mediated gene targeting has greatly contributed to genetic analysis in a wide range of species, but is highly inefficient in human cells because of overwhelmingly frequent random integration events, whose molecular mechanism remains elusive. Here we show that DNA polymerase θ, despite its minor role in chromosomal DNA repair, substantially contributes to random integration, and that cells lacking both DNA polymerase θ and DNA ligase IV, which is essential for non-homologous end joining (NHEJ), exhibit 100% efficiency of spontaneous gene targeting by virtue of undetectable levels of random integration. Thus, DNA polymerase θ-mediated end joining is the sole homology-independent repair route in the absence of NHEJ and, intriguingly, their combined absence reveals rare Alu-Alu recombination events utilizing a stretch of homology. Our findings provide new insights into the mechanics of foreign DNA integration and the role of DNA polymerase θ in human genome maintenance.
Collapse
Affiliation(s)
- Shinta Saito
- Department of Life and Environmental System Science, Graduate School of Nanobioscience, Yokohama City University, Yokohama 236-0027, Japan
| | - Ryo Maeda
- Department of Biology, Graduate School of Science, Chiba University, Chiba 263-8522, Japan
| | - Noritaka Adachi
- Department of Life and Environmental System Science, Graduate School of Nanobioscience, Yokohama City University, Yokohama 236-0027, Japan
- Advanced Medical Research Center, Yokohama City University, Yokohama 236-0004, Japan
| |
Collapse
|
|
18
|
|
|
19
|
Lee HB, Sundberg BN, Sigafoos AN, Clark KJ. Genome Engineering with TALE and CRISPR Systems in Neuroscience. Front Genet 2016; 7:47. [PMID: 27092173 PMCID: PMC4821859 DOI: 10.3389/fgene.2016.00047] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2016] [Accepted: 03/16/2016] [Indexed: 12/26/2022] Open
Abstract
Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience.
Collapse
Affiliation(s)
- Han B Lee
- Neurobiology of Disease Graduate Program, Mayo Graduate School Rochester, MN, USA
| | - Brynn N Sundberg
- Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, MN, USA
| | - Ashley N Sigafoos
- Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, MN, USA
| | - Karl J Clark
- Neurobiology of Disease Graduate Program, Mayo Graduate SchoolRochester, MN, USA; Department of Biochemistry and Molecular Biology, Mayo ClinicRochester, MN, USA
| |
Collapse
|
|
20
|
Aravalli RN, Steer CJ. Gene editing technology as an approach to the treatment of liver diseases. Expert Opin Biol Ther 2016; 16:595-608. [PMID: 26914853 DOI: 10.1517/14712598.2016.1158808] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
|
|
21
|
Xia G, Gao Y, Jin S, Subramony SH, Terada N, Ranum LPW, Swanson MS, Ashizawa T. Genome modification leads to phenotype reversal in human myotonic dystrophy type 1 induced pluripotent stem cell-derived neural stem cells. Stem Cells 2016; 33:1829-38. [PMID: 25702800 DOI: 10.1002/stem.1970] [Citation(s) in RCA: 48] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2014] [Accepted: 01/17/2015] [Indexed: 12/15/2022]
Abstract
Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats in the 3'-untranslated region (3' UTR) of the DMPK gene. Correcting the mutation in DM1 stem cells would be an important step toward autologous stem cell therapy. The objective of this study is to demonstrate in vitro genome editing to prevent production of toxic mutant transcripts and reverse phenotypes in DM1 stem cells. Genome editing was performed in DM1 neural stem cells (NSCs) derived from human DM1 induced pluripotent stem (iPS) cells. An editing cassette containing SV40/bGH polyA signals was integrated upstream of the CTG repeats by TALEN-mediated homologous recombination (HR). The expression of mutant CUG repeats transcript was monitored by nuclear RNA foci, the molecular hallmarks of DM1, using RNA fluorescence in situ hybridization. Alternative splicing of microtubule-associated protein tau (MAPT) and muscleblind-like (MBNL) proteins were analyzed to further monitor the phenotype reversal after genome modification. The cassette was successfully inserted into DMPK intron 9 and this genomic modification led to complete disappearance of nuclear RNA foci. MAPT and MBNL 1, 2 aberrant splicing in DM1 NSCs were reversed to normal pattern in genome-modified NSCs. Genome modification by integration of exogenous polyA signals upstream of the DMPK CTG repeat expansion prevents the production of toxic RNA and leads to phenotype reversal in human DM1 iPS-cells derived stem cells. Our data provide proof-of-principle evidence that genome modification may be used to generate genetically modified progenitor cells as a first step toward autologous cell transfer therapy for DM1.
Collapse
Affiliation(s)
- Guangbin Xia
- Department of Neurology, University of Florida, College of Medicine, Gainesville, Florida, USA.,Center for Cellular Reprogramming, University of Florida, College of Medicine, Gainesville, Florida, USA.,Center for NeuroGenetics, University of Florida, College of Medicine, Gainesville, Florida, USA.,The Evelyn L & William F. McKnight Brain Institute, University of Florida, Gainesville, Florida, USA.,Department of Neuroscience, University of Florida, Gainesville, Florida, USA
| | - Yuanzheng Gao
- Department of Neurology, University of Florida, College of Medicine, Gainesville, Florida, USA.,The Evelyn L & William F. McKnight Brain Institute, University of Florida, Gainesville, Florida, USA
| | - Shouguang Jin
- Department of Molecular Genetics and Microbiology, College of Medicine, Gainesville, Florida, USA
| | - S H Subramony
- Department of Neurology, University of Florida, College of Medicine, Gainesville, Florida, USA.,Center for NeuroGenetics, University of Florida, College of Medicine, Gainesville, Florida, USA.,The Evelyn L & William F. McKnight Brain Institute, University of Florida, Gainesville, Florida, USA
| | - Naohiro Terada
- Center for Cellular Reprogramming, University of Florida, College of Medicine, Gainesville, Florida, USA.,Department of Pathology, Immunology & Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida, USA
| | - Laura P W Ranum
- Department of Neurology, University of Florida, College of Medicine, Gainesville, Florida, USA.,Center for NeuroGenetics, University of Florida, College of Medicine, Gainesville, Florida, USA.,Department of Molecular Genetics and Microbiology, College of Medicine, Gainesville, Florida, USA.,Genetics Institute, University of Florida, Gainesville, Florida, USA
| | - Maurice S Swanson
- Center for NeuroGenetics, University of Florida, College of Medicine, Gainesville, Florida, USA.,Department of Molecular Genetics and Microbiology, College of Medicine, Gainesville, Florida, USA.,Genetics Institute, University of Florida, Gainesville, Florida, USA
| | - Tetsuo Ashizawa
- Department of Neurology, University of Florida, College of Medicine, Gainesville, Florida, USA.,Center for Cellular Reprogramming, University of Florida, College of Medicine, Gainesville, Florida, USA.,Center for NeuroGenetics, University of Florida, College of Medicine, Gainesville, Florida, USA.,The Evelyn L & William F. McKnight Brain Institute, University of Florida, Gainesville, Florida, USA
| |
Collapse
|
|
22
|
Song YZ, Han FJ, Liu M, Xia CC, Shi WY, Dong LH. Association between Single Nucleotide Polymorphisms in XRCC3 and Radiation-Induced Adverse Effects on Normal Tissue: A Meta-Analysis. PLoS One 2015; 10:e0130388. [PMID: 26091483 PMCID: PMC4474802 DOI: 10.1371/journal.pone.0130388] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2015] [Accepted: 05/20/2015] [Indexed: 01/26/2023] Open
Abstract
The X-ray repair cross-complementing group 3 (XRCC3) protein plays an important role in the repair of DNA double-strand breaks. The relationship between XRCC3 polymorphisms and the risk of radiation-induced adverse effects on normal tissue remains inconclusive. Thus, we performed a meta-analysis to elucidate the association between XRCC3 polymorphisms and radiation-induced adverse effects on normal tissue. All eligible studies up to December 2014 were identified through a search of the PubMed, Embase and Web of Science databases. Seventeen studies involving 656 cases and 2193 controls were ultimately included in this meta-analysis. The pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated to evaluate the association between XRCC3 polymorphisms and the risk of radiation-induced normal tissue adverse effects. We found that the XRCC3 p.Thr241Met (rs861539) polymorphism was significantly associated with early adverse effects induced by radiotherapy (OR = 1.99, 95%CI: 1.31-3.01, P = 0.001). A positive association lacking statistical significance with late adverse effects was also identified (OR = 1.28, 95%CI: 0.97-1.68, P = 0.08). In addition, the rs861539 polymorphism was significantly correlated with a higher risk of adverse effects induced by head and neck area irradiation (OR = 2.41, 95%CI: 1.49-3.89, p = 0.0003) and breast irradiation (OR = 1.41, 95%CI: 1.02-1.95, p = 0.04), whereas the correlation was not significant for lung irradiation or pelvic irradiation. Furthermore, XRCC3 rs1799794 polymorphism may have a protective effect against late adverse effects induced by radiotherapy (OR = 0.47, 95%CI: 0.26-0.86, P = 0.01). Well-designed large-scale clinical studies are required to further validate our results.
Collapse
Affiliation(s)
- Yu-Zhe Song
- Department of Radiation Oncology, the First Hospital of Jilin University, Changchun, Jilin, China
| | - Fu-Jun Han
- Cancer Center, the First Hospital of Jilin University, Changchun, Jilin, China
| | - Min Liu
- Department of Radiation Oncology, the First Hospital of Jilin University, Changchun, Jilin, China
| | - Cheng-Cheng Xia
- Department of Radiation Oncology, the First Hospital of Jilin University, Changchun, Jilin, China
| | - Wei-Yan Shi
- Department of Radiation Oncology, the First Hospital of Jilin University, Changchun, Jilin, China
| | - Li-Hua Dong
- Department of Radiation Oncology, the First Hospital of Jilin University, Changchun, Jilin, China
- * E-mail:
| |
Collapse
|
|
23
|
Aravalli RN, Belcher JD, Steer CJ. Liver-targeted gene therapy: Approaches and challenges. Liver Transpl 2015; 21:718-37. [PMID: 25824605 PMCID: PMC9353592 DOI: 10.1002/lt.24122] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/07/2014] [Revised: 03/06/2015] [Accepted: 03/14/2015] [Indexed: 12/15/2022]
Abstract
The liver plays a major role in many inherited and acquired genetic disorders. It is also the site for the treatment of certain inborn errors of metabolism that do not directly cause injury to the liver. The advancement of nucleic acid-based therapies for liver maladies has been severely limited because of the myriad untoward side effects and methodological limitations. To address these issues, research efforts in recent years have been intensified toward the development of targeted gene approaches using novel genetic tools, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats as well as various nonviral vectors such as Sleeping Beauty transposons, PiggyBac transposons, and PhiC31 integrase. Although each of these methods uses a distinct mechanism of gene modification, all of them are dependent on the efficient delivery of DNA and RNA molecules into the cell. This review provides an overview of current and emerging therapeutic strategies for liver-targeted gene therapy and gene repair.
Collapse
Affiliation(s)
- Rajagopal N. Aravalli
- Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 54455
| | - John D. Belcher
- Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 54455
| | - Clifford J. Steer
- Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 54455,Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 54455
| |
Collapse
|
|
24
|
Li Z, Zeng F, Meng F, Xu Z, Zhang X, Huang X, Tang F, Gao W, Shi J, He X, Liu D, Wang C, Urschitz J, Moisyadi S, Wu Z. Generation of transgenic pigs by cytoplasmic injection of piggyBac transposase-based pmGENIE-3 plasmids. Biol Reprod 2014; 90:93. [PMID: 24671876 DOI: 10.1095/biolreprod.113.116905] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.
Collapse
Affiliation(s)
- Zicong Li
- Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, People's Republic of China
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
25
|
Magin S, Saha J, Wang M, Mladenova V, Coym N, Iliakis G. Lipofection and nucleofection of substrate plasmid can generate widely different readings of DNA end-joining efficiency in different cell lines. DNA Repair (Amst) 2013; 12:148-60. [PMID: 23286905 DOI: 10.1016/j.dnarep.2012.11.008] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2011] [Revised: 10/05/2012] [Accepted: 11/21/2012] [Indexed: 10/27/2022]
Abstract
In vivo plasmid end-joining assays are valuable tools for dissecting important qualitative and quantitative aspects of non-homologous end-joining (NHEJ)--a key mechanism for the repair of DNA double-strand breaks (DSBs) in higher eukaryotes. They enable the use of defined DNA ends as substrates for end-joining and the analysis by sequencing of the resulting junctions to identify the repair pathways engaged. Yet, plasmid assays have generated divergent results of end-joining capacity in the same DSB repair mutants when used under different conditions, which implies contributions from undefined and therefore uncontrolled parameters. To help standardize these assays, we searched for parameters underpinning these variations and identified transfection method as an important determinant. Here, we compare a lipid-based transfection method, lipofection, with an electroporation method, nucleofection, and find large, unanticipated and cell line-dependent differences in percent end-joining without recognizable trends. For example, in rodent cells, transfection using lipofection gives nearly WT end-joining in DNA-PKcs mutants and only mildly inhibited end-joining in Lig4 and Ku mutants. In contrast, transfection using nucleofection shows marked end-joining inhibition in all NHEJ mutants tested as compared to the WT. In human HCT116 cells, end-joining after nucleofection is strongly suppressed even in the WT and the differences to the mutants are small. After lipofection, in contrast, end-joining is high in WT cells and markedly suppressed in the mutants. We conclude that better understanding and control of the physicochemical/biological and analytical parameters underpinning these differences will be required to generate with plasmid assays results with quantitative power comparable to that of well-established methods of DSB analysis such as pulsed-field gel electrophoresis or γ-H2AX foci scoring. Until then, caution is needed in the interpretation of the results obtained - particularly with reference to pathway efficiency and residual damage - and confirmation of critical results with alternative transfection approaches is advisable.
Collapse
Affiliation(s)
- Simon Magin
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany
| | | | | | | | | | | |
Collapse
|
|
26
|
Huang JT, Dooner HK. The spectrum and frequency of self-inflicted and host gene mutations produced by the transposon Ac in maize. THE PLANT CELL 2012; 24:4149-4162. [PMID: 23110898 PMCID: PMC3517242 DOI: 10.1105/tpc.112.104265] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/20/2012] [Revised: 09/21/2012] [Accepted: 10/09/2012] [Indexed: 05/27/2023]
Abstract
The autonomous transposon Activator (Ac) is a powerful mutagen. Ac-induced mutations range from small footprints of host sequences to large rearrangements of transposon or host sequences. These mutations arise by different repair mechanisms of the double-strand break produced by Ac excision: footprints by nonhomologous end joining and rearrangements by various mechanisms, including DNA replication repair. Footprints greatly outnumber other mutations, masking them because they usually share a nonfunctional phenotype. To determine the spectrum and frequencies of host and self-mutations generated by Ac, we used an allele harboring Ac in the 5' untranslated region bronze (bz). In this system, simple excisions produce purple revertants, whereas deletions of host or transposon sequences produce stable bronze (bz-s) mutants. Internal and terminal deletions of Ac predominated among the 72 bz-s derivatives. Most internal deletions (52 of 54) behaved as nonautonomous Dissociation (Ds) elements. All nine terminal deletions or fractured Ac (fAc) elements had rearrangements of adjacent host sequences. Most Ds and fAc deletion junctions displayed microhomologies and contained filler DNA from nearby sequences, suggesting an origin by DNA repair synthesis followed by microhomology-mediated end joining. All mutations occurred more frequently in pollen, where one in 200 grains carried new Ds or fAc elements.
Collapse
Affiliation(s)
- Jun T. Huang
- Waksman Institute, Rutgers University, Piscataway, New Jersey 08854
- Department of Plant Biology, Rutgers University, New Brunswick, New Jersey 08901
| | - Hugo K. Dooner
- Waksman Institute, Rutgers University, Piscataway, New Jersey 08854
- Department of Plant Biology, Rutgers University, New Brunswick, New Jersey 08901
| |
Collapse
|
|
27
|
A novel approach for the rapid mutagenesis and directed evolution of the structural genes of west nile virus. J Virol 2012; 86:3501-12. [PMID: 22258236 DOI: 10.1128/jvi.06435-11] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Molecular clone technology has proven to be a powerful tool for investigating the life cycle of flaviviruses, their interactions with the host, and vaccine development. Despite the demonstrated utility of existing molecular clone strategies, the feasibility of employing these existing approaches in large-scale mutagenesis studies is limited by the technical challenges of manipulating relatively large molecular clone plasmids that can be quite unstable when propagated in bacteria. We have developed a novel strategy that provides an extremely rapid approach for the introduction of mutations into the structural genes of West Nile virus (WNV). The backbone of this technology is a truncated form of the genome into which DNA fragments harboring the structural genes are ligated and transfected directly into mammalian cells, bypassing entirely the requirement for cloning in bacteria. The transfection of cells with this system results in the rapid release of WNV that achieves a high titer (∼10(7) infectious units/ml in 48 h). The suitability of this approach for large-scale mutagenesis efforts was established in two ways. First, we constructed and characterized a library of variants encoding single defined amino acid substitutions at the 92 residues of the "pr" portion of the precursor-to-membrane (prM) protein. Analysis of a subset of these variants identified a mutation that conferred resistance to neutralization by an envelope protein-specific antibody. Second, we employed this approach to accelerate the identification of mutations that allow escape from neutralizing antibodies. Populations of WNV encoding random changes in the E protein were produced in the presence of a potent monoclonal antibody, E16. Viruses resistant to neutralization were identified in a single passage. Together, we have developed a simple and rapid approach to produce infectious WNV that accelerates the process of manipulating the genome to study the structure and function of the structural genes of this important human pathogen.
Collapse
|
|
28
|
Sharma S, Choudhary B, Raghavan SC. Efficiency of nonhomologous DNA end joining varies among somatic tissues, despite similarity in mechanism. Cell Mol Life Sci 2011; 68:661-76. [PMID: 20680388 PMCID: PMC11114952 DOI: 10.1007/s00018-010-0472-x] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2010] [Revised: 07/07/2010] [Accepted: 07/16/2010] [Indexed: 01/09/2023]
Abstract
Failure to repair DNA double-strand breaks (DSBs) can lead to cell death or cancer. Although nonhomologous end joining (NHEJ) has been studied extensively in mammals, little is known about it in primary tissues. Using oligomeric DNA mimicking endogenous DSBs, NHEJ in cell-free extracts of rat tissues were studied. Results show that efficiency of NHEJ is highest in lungs compared to other somatic tissues. DSBs with compatible and blunt ends joined without modifications, while noncompatible ends joined with minimal alterations in lungs and testes. Thymus exhibited elevated joining, followed by brain and spleen, which could be correlated with NHEJ gene expression. However, NHEJ efficiency was poor in terminally differentiated organs like heart, kidney and liver. Strikingly, NHEJ junctions from these tissues also showed extensive deletions and insertions. Hence, for the first time, we show that despite mode of joining being generally comparable, efficiency of NHEJ varies among primary tissues of mammals.
Collapse
Affiliation(s)
- Sheetal Sharma
- Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012 India
| | - Bibha Choudhary
- Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012 India
- Manipal Institute of Regenerative Medicine, Manipal University, Bangalore, 560 071 India
| | - Sathees C. Raghavan
- Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012 India
| |
Collapse
|
|
29
|
Dai J, Cui X, Zhu Z, Hu W. Non-homologous end joining plays a key role in transgene concatemer formation in transgenic zebrafish embryos. Int J Biol Sci 2010; 6:756-68. [PMID: 21152116 PMCID: PMC2999851 DOI: 10.7150/ijbs.6.756] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2010] [Accepted: 11/28/2010] [Indexed: 01/11/2023] Open
Abstract
This study focused on concatemer formation and integration pattern of transgenes in zebrafish embryos. A reporter plasmid based on enhanced green fluorescent protein (eGFP) driven by Cytomegalovirus (CMV) promoter, pCMV-pax6in-eGFP, was constructed to reflect transgene behavior in the host environment. After removal of the insertion fragment by double digestion with various combinations of restriction enzymes, linearized pCMV-pax6in-eGFP vectors were generated with different combinations of 5′-protruding, 3′-protruding, and blunt ends that were microinjected into zebrafish embryos. Repair of double-strand breaks (DSBs) was monitored by GFP expression following religation of the reporter gene. One-hundred-and-ninety-seven DNA fragments were amplified from GFP-positive embryos and sequenced to analyze the repair characteristics of different DSB end combinations. DSBs involving blunt and asymmetric protruding ends were repaired efficiently by direct ligation of blunt ends, ligation after blunting and fill-in, or removed by cutting. Repair of DSBs with symmetric 3′-3′ protrusions was less efficient and utilized template-directed repair. The results suggest that non-homologous end joining (NHEJ) was the principal mechanism of exogenous gene concatemer formation and integration of transgenes into the genome of transgenic zebrafish.
Collapse
Affiliation(s)
- Jun Dai
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
| | | | | | | |
Collapse
|
|
30
|
Sharma S, Raghavan SC. Nonhomologous DNA end joining in cell-free extracts. J Nucleic Acids 2010; 2010. [PMID: 20936167 PMCID: PMC2945661 DOI: 10.4061/2010/389129] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2010] [Revised: 07/14/2010] [Accepted: 08/05/2010] [Indexed: 12/19/2022] Open
Abstract
Among various DNA damages, double-strand breaks (DSBs) are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.
Collapse
Affiliation(s)
- Sheetal Sharma
- Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
| | | |
Collapse
|
|
31
|
Toyoda E, Kurosawa A, Kamekawa H, Adachi N. Topoisomerase IIα inhibition following DNA transfection greatly enhances random integration in a human pre-B lymphocyte cell line. Biochem Biophys Res Commun 2009; 382:492-6. [DOI: 10.1016/j.bbrc.2009.03.047] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2009] [Accepted: 03/02/2009] [Indexed: 11/15/2022]
|
|
32
|
Iiizumi S, Kurosawa A, So S, Ishii Y, Chikaraishi Y, Ishii A, Koyama H, Adachi N. Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting. Nucleic Acids Res 2008; 36:6333-42. [PMID: 18835848 PMCID: PMC2577324 DOI: 10.1093/nar/gkn649] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
In higher animal cells, the principal limitation of gene-targeting technology is the extremely low efficiency of targeted integration, which occurs three to four orders of magnitude less frequently than random integration. Assuming that random integration mechanistically involves non-homologous end-joining (NHEJ), inactivation of this pathway should reduce random integration and may enhance gene targeting. To test this possibility, we examined the frequencies of random and targeted integration in NHEJ-deficient chicken DT40 and human Nalm-6 cell lines. As expected, loss of NHEJ resulted in drastically reduced random integration in DT40 cells. Unexpectedly, however, this was not the case for Nalm-6 cells, indicating that NHEJ is not the sole mechanism of random integration. Nevertheless, we present evidence that NHEJ inactivation can lead to enhanced gene targeting through a reduction of random integration and/or an increase in targeted integration by homologous recombination. Most intriguingly, our results show that, in the absence of functional NHEJ, random integration of targeting vectors occurs more frequently than non-targeting vectors (harboring no or little homology to the host genome), implying that suppression of NHEJ-independent random integration events is needed to greatly enhance gene targeting in animal cells.
Collapse
Affiliation(s)
- Susumu Iiizumi
- International Graduate School of Arts and Sciences and Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan
| | | | | | | | | | | | | | | |
Collapse
|
|
33
|
Shrivastav M, De Haro LP, Nickoloff JA. Regulation of DNA double-strand break repair pathway choice. Cell Res 2008; 18:134-47. [PMID: 18157161 DOI: 10.1038/cr.2007.111] [Citation(s) in RCA: 951] [Impact Index Per Article: 55.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1(XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.
Collapse
Affiliation(s)
- Meena Shrivastav
- Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine and Cancer Center, Albuquerque, NM 87131, USA
| | | | | |
Collapse
|
|
34
|
Odom GL, Gregorevic P, Chamberlain JS. Viral-mediated gene therapy for the muscular dystrophies: successes, limitations and recent advances. BIOCHIMICA ET BIOPHYSICA ACTA 2007; 1772:243-62. [PMID: 17064882 PMCID: PMC1894910 DOI: 10.1016/j.bbadis.2006.09.007] [Citation(s) in RCA: 81] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/20/2006] [Revised: 09/16/2006] [Accepted: 09/20/2006] [Indexed: 02/07/2023]
Abstract
Much progress has been made over the past decade elucidating the molecular basis for a variety of muscular dystrophies (MDs). Accordingly, there are examples of mouse models of MD whose disease progression has been halted in large part with the use of viral vector technology. Even so, we must acknowledge significant limitations of present vector systems that must be overcome prior to successful treatment of humans with such approaches. This review will present a variety of viral-mediated therapeutic strategies aimed at counteracting the muscle-wasting symptoms associated with muscular dystrophy. We include viral vector systems used for muscle gene transfer, with a particular emphasis on adeno-associated virus. Findings of several encouraging studies focusing on repair of the mutant dystrophin gene are also included. Lastly, we present a discussion of muscle compensatory therapeutics being considered that include pathways involved in the up-regulation of utrophin, promotion of cellular adhesion, enhancement of muscle mass, and antagonism of the inflammatory response. Considering the complexity of the muscular dystrophies, it appears likely that a multilayered approach tailored to a patient sub-group may be warranted in order to effectively contest the progression of this devastating disease.
Collapse
Affiliation(s)
- Guy L. Odom
- Department of Neurology Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, 1959 NE Pacific Street, Seattle, WA, 98195-7720, USA
| | - Paul Gregorevic
- Department of Neurology Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, 1959 NE Pacific Street, Seattle, WA, 98195-7720, USA
| | - Jeffrey S. Chamberlain
- Department of Neurology Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, 1959 NE Pacific Street, Seattle, WA, 98195-7720, USA
| |
Collapse
|
|
35
|
Abstract
DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages.
Collapse
|
|
36
|
Kegel A, Martinez P, Carter SD, Åström SU. Genome wide distribution of illegitimate recombination events in Kluyveromyces lactis. Nucleic Acids Res 2006; 34:1633-45. [PMID: 16549875 PMCID: PMC1405753 DOI: 10.1093/nar/gkl064] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2005] [Revised: 01/21/2006] [Accepted: 02/28/2006] [Indexed: 11/13/2022] Open
Abstract
Illegitimate recombination (IR) is the process by which two DNA molecules not sharing homology to each other are joined. In Kluyveromyces lactis, integration of heterologous DNA occurred very frequently therefore constituting an excellent model organism to study IR. IR was completely dependent on the nonhomologous end-joining (NHEJ) pathway for DNA double strand break (DSB) repair and we detected no other pathways capable of mediating IR. NHEJ was very versatile, capable of repairing both blunt and non-complementary ends efficiently. Mapping the locations of genomic IR-events revealed target site preferences, in which intergenic regions (IGRs) and ribosomal DNA were overrepresented six-fold compared to open reading frames (ORFs). The IGR-events occurred predominantly within transcriptional regulatory regions. In a rad52 mutant strain IR still preferentially occurred at IGRs, indicating that DSBs in ORFs were not primarily repaired by homologous recombination (HR). Introduction of ectopic DSBs resulted in the efficient targeting of IR to these sites, strongly suggesting that IR occurred at spontaneous mitotic DSBs. The targeting efficiency was equal when ectopic breaks were introduced in an ORF or an IGR. We propose that spontaneous DSBs arise more frequently in transcriptional regulatory regions and in rDNA and such DSBs can be mapped by analyzing IR target sites.
Collapse
Affiliation(s)
- Andreas Kegel
- Department of Developmental Biology, Wennergren Institute, Stockholm UniversitySE-106 91 Stockholm, Sweden
| | - Paula Martinez
- Department of Developmental Biology, Wennergren Institute, Stockholm UniversitySE-106 91 Stockholm, Sweden
| | - Sidney D. Carter
- Department of Developmental Biology, Wennergren Institute, Stockholm UniversitySE-106 91 Stockholm, Sweden
| | - Stefan U. Åström
- Department of Developmental Biology, Wennergren Institute, Stockholm UniversitySE-106 91 Stockholm, Sweden
| |
Collapse
|
|
37
|
Pagamjav O, Sakata T, Matsumura T, Yamaguchi T, Fukushi H. Natural recombinant between equine herpesviruses 1 and 4 in the ICP4 gene. Microbiol Immunol 2005; 49:167-79. [PMID: 15722602 DOI: 10.1111/j.1348-0421.2005.tb03716.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Equine herpesvirus 1 (EHV-1) is a pathogen causing rhinopneumonia in young horses, abortion in mares, and myeloencephalitis in adult horses. Two types, EHV-1 P and EHV-1 B, have recently been dominant among 16 electropherotypes. EHV-1 P and EHV-1 B viruses were compared by long and accurate polymerase chain reaction (LA-PCR) and restriction fragment length polymorphism (RFLP) analysis. Differences in restriction sites were found to be focused in ORF64, which encodes the infected cell protein 4 (ICP4), and downstream of the ICP4 gene. The 3 ' -end and downstream of ICP4 gene of EHV-1 B were found to be replaced by the corresponding region of EHV-4, indicating that EHV-1 B is a naturally occurring recombinant virus between progenitors of EHV-1 P and EHV-4. This is the first report showing a natural interspecies recombinant in alphaherpesviruses.
Collapse
Affiliation(s)
- Ochir Pagamjav
- Department of Applied Veterinary Sciences, United Graduate School of Veterinary Sciences, Gifu University, Japan
| | | | | | | | | |
Collapse
|
|
38
|
Norberg P, Bergström T, Rekabdar E, Lindh M, Liljeqvist JA. Phylogenetic analysis of clinical herpes simplex virus type 1 isolates identified three genetic groups and recombinant viruses. J Virol 2004; 78:10755-64. [PMID: 15367642 PMCID: PMC516408 DOI: 10.1128/jvi.78.19.10755-10764.2004] [Citation(s) in RCA: 141] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen which establishes lifelong infections. In the present study, we determined the sequence diversity of the complete genes coding for glycoproteins G (gG), I (gI), and E (gE), comprising 2.3% of the HSV-1 genome and located within the unique short (US) region, for 28 clinical HSV-1 isolates inducing oral lesions, genital lesions, or encephalitis. Laboratory strains F and KOS321 were sequenced in parallel. Phylogenetic analysis, including analysis of laboratory strain 17 (GenBank), revealed that the sequences were separated into three genetic groups. The identification of different genogroups facilitated the detection of recombinant viruses by using specific nucleotide substitutions as recombination markers. Seven of the isolates and strain 17 displayed sequences consistent with intergenic recombination, and at least four isolates were intragenic recombinants. The observed frequency of recombination based on an analysis of a short stretch of the US region suggests that most full-length HSV-1 genomes consist of a mosaic of segments from different genetic groups. Polymorphic tandem repeat regions, consisting of two to eight blocks of 21 nucleotides in the gI gene and seven to eight repeats of 3 nucleotides in the gG gene, were also detected. Laboratory strain KOS321 displayed a frameshift mutation in the gI gene with a subsequent alteration of the deduced intracellular portion of the protein. The presence of polymorphic tandem repeat regions and the different genogroup identities can be used for molecular epidemiology studies and for further detection of recombination in the HSV-1 genome.
Collapse
Affiliation(s)
- Peter Norberg
- Department of Virology, University of Göteborg, Guldhedsgatan 10 B, S-413 46 Göteborg, Sweden.
| | | | | | | | | |
Collapse
|
|
39
|
Lee GS, Neiditch MB, Salus SS, Roth DB. RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination. Cell 2004; 117:171-84. [PMID: 15084256 DOI: 10.1016/s0092-8674(04)00301-0] [Citation(s) in RCA: 199] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2003] [Revised: 01/30/2004] [Accepted: 02/12/2004] [Indexed: 11/28/2022]
Abstract
The two major pathways for repairing double-strand breaks (DSBs), homologous recombination and nonhomologous end joining (NHEJ), have traditionally been thought to operate in different stages of the cell cycle. This division of labor is not absolute, however, and precisely what governs the choice of pathway to repair a given DSB has remained enigmatic. We pursued this question by studying the site-specific DSBs created during V(D)J recombination, which relies on classical NHEJ to repair the broken ends. We show that mutations that form unstable RAG postcleavage complexes allow DNA ends to participate in both homologous recombination and the error-prone alternative NHEJ pathway. By abrogating a key function of the complex, these mutations reveal it to be a molecular shepherd that guides DSBs to the proper pathway. We also find that RAG-mediated nicks efficiently stimulate homologous recombination and discuss the implications of these findings for oncogenic chromosomal rearrangements, evolution, and gene targeting.
Collapse
Affiliation(s)
- Gregory S Lee
- The Skirball Institute of Biomolecular Medicine, Lab 2-10 and Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
| | | | | | | |
Collapse
|
|
40
|
West SC, Chappell C, Hanakahi LA, Masson JY, McIlwraith MJ, Van Dyck E. Double-strand break repair in human cells. COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 2003; 65:315-21. [PMID: 12760045 DOI: 10.1101/sqb.2000.65.315] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Affiliation(s)
- S C West
- Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD, United Kingdom
| | | | | | | | | | | |
Collapse
|
|
41
|
Przewloka MR, Pardington PE, Yannone SM, Chen DJ, Cary RB. In vitro and in vivo interactions of DNA ligase IV with a subunit of the condensin complex. Mol Biol Cell 2003; 14:685-97. [PMID: 12589063 PMCID: PMC150001 DOI: 10.1091/mbc.e01-11-0117] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Several findings have revealed a likely role for DNA ligase IV, and interacting protein XRCC4, in the final steps of mammalian DNA double-strand break repair. Recent evidence suggests that the human DNA ligase IV protein plays a critical role in the maintenance of genomic stability. To identify protein-protein interactions that may shed further light on the molecular mechanisms of DSB repair and the biological roles of human DNA ligase IV, we have used the yeast two-hybrid system in conjunction with traditional biochemical methods. These efforts have resulted in the identification of a physical association between the DNA ligase IV polypeptide and the human condensin subunit known as hCAP-E. The hCAP-E polypeptide, a member of the Structural Maintenance of Chromosomes (SMC) super-family of proteins, coimmunoprecipitates from cell extracts with DNA ligase IV. Immunofluorescence studies reveal colocalization of DNA ligase IV and hCAP-E in the interphase nucleus, whereas mitotic cells display colocalization of both polypeptides on mitotic chromosomes. Strikingly, the XRCC4 protein is excluded from the area of mitotic chromosomes, suggesting the formation of specialized DNA ligase IV complexes subject to cell cycle regulation. We discuss our findings in light of known and hypothesized roles for ligase IV and the condensin complex.
Collapse
Affiliation(s)
- Marcin R Przewloka
- Los Alamos National Laboratory, Biosciences Division, New Mexico 87545, USA
| | | | | | | | | |
Collapse
|
|
42
|
Frank-Vaillant M, Marcand S. Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination. Mol Cell 2002; 10:1189-99. [PMID: 12453425 DOI: 10.1016/s1097-2765(02)00705-0] [Citation(s) in RCA: 152] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The stability of DNA ends generated by the HO endonuclease in yeast is surprisingly high with a half-life of more than an hour. This transient stability is unaffected by mutations that abolish nonhomologous end joining (NHEJ). The unprocessed ends interact with Yku70p and Yku80p, two proteins required for NHEJ, but not significantly with Rad52p, a protein involved in homologous recombination (HR). Repair of a double-strand break by NHEJ is unaffected by the possibility of HR, although the use of HR is increased in NHEJ-defective cells. Partial in vitro 5' strand processing suppresses NHEJ but not HR. These results show that NHEJ precedes HR temporally, and that the availability of substrate dictates the particular pathway used. We propose that transient stability of DNA ends is a foundation for the permanent stability of telomeres.
Collapse
Affiliation(s)
- Marie Frank-Vaillant
- Laboratoire du Cycle Cellulaire, Service de Biochimie et de Génétique Moléculaire, CEA/Saclay, 91191 Gif sur Yvette Cedex, France
| | | |
Collapse
|
|
43
|
Zeng ZC, Jiang GL, Wang GM, Tang ZY, Curran WJ, Iliakis G. DNA-PKcs subunits in radiosensitization by hyperthermia on hepatocellular carcinoma hepG2 cell line. World J Gastroenterol 2002; 8:797-803. [PMID: 12378618 PMCID: PMC4656564 DOI: 10.3748/wjg.v8.i5.797] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the role of DNA-PKcs subunits in radiosensitization by hyperthermia on hepatocellular carcinoma HepG2 cell lines.
METHODS: HepG2 cells were exposed to hyperthermia and irradiation. Hyperthermia was given at 45.5 °C. Cell survival was determined by an in vitro clonogenic assay for the cells treated with or without hyperthermia at various time points. DNA DSB rejoining was measured using asymmetric field inversion gel electrophoresis (AFIGE). The DNA-PKcs activities were measured using DNA-PKcs enzyme assay system.
RESULTS: Hyperthermia can significantly enhance irradiation-killing cells. Thermal enhancement ratio as calculated at 10% survival was 2.02. The difference in radiosensitivity between two treatment modes manifested as a difference in the α components and the almost same β components, which α value was considerably higher in the cells of combined radiation and hyperthermia as compared with irradiating cells (1.07 Gy-1vs 0.44 Gy-1). Survival fraction showed 1 logarithm increase after an 8-hour interval between heat and irradiation, whereas DNA-PKcs activity did not show any recovery. The cells were exposed to heat 5 min only, DNA-PKcs activity was inhibited at the nadir, even though the exposure time was lengthened. Whereas the ability of DNA DSB rejoining was inhibited with the increase of the length of hyperthermic time. The repair kinetics of DNA DSB rejoining after treatment with Wortmannin is different from the hyperthermic group due to the striking high slow rejoining component.
CONCLUSION: Determination with the cell extracts and the peptide phosphorylation assay, DNA-PKcs activity was inactivated by heat treatment at 45.5 °C, and could not restore. Cell survival is not associated with the DNA-PKcs inactivity after heat. DNA-PKcs is not a unique factor affecting the DNA DSB repair. This suggests that DNA-PKcs do not play a crucial role in the enhancement of cellular radiosensitivity by hyperthermia.
Collapse
Affiliation(s)
- Zhao-Chong Zeng
- Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
| | | | | | | | | | | |
Collapse
|
|
44
|
Brenneman MA, Wagener BM, Miller CA, Allen C, Nickoloff JA. XRCC3 controls the fidelity of homologous recombination: roles for XRCC3 in late stages of recombination. Mol Cell 2002; 10:387-95. [PMID: 12191483 DOI: 10.1016/s1097-2765(02)00595-6] [Citation(s) in RCA: 148] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
XRCC3 is a RAD51 paralog that functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). XRCC3 mutation causes severe chromosome instability. We find that XRCC3 mutant cells display radically altered HR product spectra, with increased gene conversion tract lengths, increased frequencies of discontinuous tracts, and frequent local rearrangements associated with HR. These results indicate that XRCC3 function is not limited to HR initiation, but extends to later stages in formation and resolution of HR intermediates, possibly by stabilizing heteroduplex DNA. The results further demonstrate that HR defects can promote genomic instability not only through failure to initiate HR (leading to nonhomologous repair) but also through aberrant processing of HR intermediates. Both mechanisms may contribute to carcinogenesis in HR-deficient cells.
Collapse
Affiliation(s)
- Mark A Brenneman
- Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
| | | | | | | | | |
Collapse
|
|
45
|
Odersky A, Panyutin IV, Panyutin IG, Schunck C, Feldmann E, Goedecke W, Neumann RD, Obe G, Pfeiffer P. Repair of sequence-specific 125I-induced double-strand breaks by nonhomologous DNA end joining in mammalian cell-free extracts. J Biol Chem 2002; 277:11756-64. [PMID: 11821407 DOI: 10.1074/jbc.m111304200] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair. Rejoining of DSB produced by decay of (125)I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the (125)I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin (125)I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the (125)I-TFO within its target site. We show that NHEJ of (125)I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after (125)I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.
Collapse
Affiliation(s)
- Andrea Odersky
- Institut für Genetik FB9, Universität Essen, Universitätsstrasse 5, D-45117 Essen, Germany
| | | | | | | | | | | | | | | | | |
Collapse
|
|
46
|
Allen C, Kurimasa A, Brenneman MA, Chen DJ, Nickoloff JA. DNA-dependent protein kinase suppresses double-strand break-induced and spontaneous homologous recombination. Proc Natl Acad Sci U S A 2002; 99:3758-63. [PMID: 11904432 PMCID: PMC122597 DOI: 10.1073/pnas.052545899] [Citation(s) in RCA: 138] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2000] [Indexed: 01/22/2023] Open
Abstract
DNA-dependent protein kinase (DNA-PK), composed of Ku70, Ku80, and the catalytic subunit (DNA-PKcs), is involved in repairing double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ). Certain proteins involved in NHEJ are also involved in DSB repair by homologous recombination (HR). To test the effects of DNA-PKcs on DSB-induced HR, we integrated neo direct repeat HR substrates carrying the I-SceI recognition sequence into DNA-PKcs-defective Chinese hamster ovary (V3) cells. The DNA-PKcs defect was complemented with a human DNA-PKcs cDNA. DSB-induced HR frequencies were 1.5- to 3-fold lower with DNA-PKcs complementation. In complemented and uncomplemented strains, all products arose by gene conversion without associated crossover, and average conversion tract lengths were similar. Suppression of DSB-induced HR in complemented cells probably reflects restoration of NHEJ, consistent with competition between HR and NHEJ during DSB repair. Interestingly, spontaneous HR rates were 1.6- to >3.5-fold lower with DNA-PKcs complementation. DNA-PKcs may suppress spontaneous HR through NHEJ of spontaneous DSBs, perhaps at stalled or blocked replication forks. Because replication protein A (RPA) is involved in both replication and HR, and is phosphorylated by DNA-PKcs, it is possible that the suppression of spontaneous HR by DNA-PKcs reflects regulation of replication-dependent HR by DNA-PKcs, perhaps by means of phosphorylation of RPA.
Collapse
Affiliation(s)
- Chris Allen
- Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
| | | | | | | | | |
Collapse
|
|
47
|
Goff SP. Intracellular trafficking of retroviral genomes during the early phase of infection: viral exploitation of cellular pathways. J Gene Med 2001; 3:517-28. [PMID: 11778899 DOI: 10.1002/1521-2254(200111)3:6<517::aid-jgm234>3.0.co;2-e] [Citation(s) in RCA: 84] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Retroviruses enter cells through specific cell-surface receptors and then embark on a journey that ultimately leads to the establishment of the integrated proviral DNA. The steps of the journey include the reverse transcription of the viral RNA into DNA, the trafficking of the viral protein-DNA complex through the cytoplasm, the entry of the complex into the nucleus, and the insertion of the linear viral DNA into the host genome. All these steps are likely to involve specific interactions of viral proteins with host machinery. Our knowledge of the details of these interactions is very limited but is rapidly expanding, and should provide a deeper understanding of the pathways and components used by the different classes of retroviruses. This knowledge in turn should enable the development of better and more efficient retroviral vectors for use in gene therapy protocols in vivo.
Collapse
Affiliation(s)
- S P Goff
- Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA.
| |
Collapse
|
|
48
|
Yannone SM, Roy S, Chan DW, Murphy MB, Huang S, Campisi J, Chen DJ. Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase. J Biol Chem 2001; 276:38242-8. [PMID: 11477099 DOI: 10.1074/jbc.m101913200] [Citation(s) in RCA: 176] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
DNA double-strand breaks (DSBs) are a highly mutagenic and potentially lethal damage that occurs in all organisms. Mammalian cells repair DSBs by homologous recombination and non-homologous end joining, the latter requiring DNA-dependent protein kinase (DNA-PK). Werner syndrome is a disorder characterized by genomic instability, aging pathologies and defective WRN, a RecQ-like helicase with exonuclease activity. We show that WRN interacts directly with the catalytic subunit of DNA-PK (DNA-PK(CS)), which inhibits both the helicase and exonuclease activities of WRN. In addition we show that WRN forms a stable complex on DNA with DNA-PK(CS) and the DNA binding subunit Ku. This assembly reverses WRN enzymatic inhibition. Finally, we show that WRN is phosphorylated in vitro by DNA-PK and requires DNA-PK for phosphorylation in vivo, and that cells deficient in WRN are mildly sensitive to ionizing radiation. These data suggest that DNA-PK and WRN may function together in DNA metabolism and implicate WRN function in non-homologous end joining.
Collapse
Affiliation(s)
- S M Yannone
- Life Sciences Division, Department of Molecular and Cellular Biology, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
| | | | | | | | | | | | | |
Collapse
|
|
49
|
Rattray AJ, McGill CB, Shafer BK, Strathern JN. Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1. Genetics 2001; 158:109-22. [PMID: 11333222 PMCID: PMC1461648 DOI: 10.1093/genetics/158.1.109] [Citation(s) in RCA: 118] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.
Collapse
Affiliation(s)
- A J Rattray
- Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA
| | | | | | | |
Collapse
|
|
50
|
Clikeman JA, Khalsa GJ, Barton SL, Nickoloff JA. Homologous recombinational repair of double-strand breaks in yeast is enhanced by MAT heterozygosity through yKU-dependent and -independent mechanisms. Genetics 2001; 157:579-89. [PMID: 11156980 PMCID: PMC1461527 DOI: 10.1093/genetics/157.2.579] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
DNA double-strand breaks (DSBs) are repaired by homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ in yeast chromosomes has been observed only when HR is blocked, as in rad52 mutants or in the absence of a homologous repair template. We detected yKu70p-dependent imprecise NHEJ at a frequency of approximately 0.1% in HR-competent Rad+ haploid cells. Interestingly, yku70 mutation increased DSB-induced HR between direct repeats by 1.3-fold in a haploid strain and by 1.5-fold in a MAT homozygous (a/a) diploid, but yku70 had no effect on HR in a MAT heterozygous (a/alpha) diploid. yku70 might increase HR because it eliminates the competing precise NHEJ (religation) pathway and/or because yKu70p interferes directly or indirectly with HR. Despite the yku70-dependent increase in a/a cells, HR remained 2-fold lower than in a/alpha cells. Cell survival was also lower in a/a cells and correlated with the reduction in HR. These results indicate that MAT heterozygosity enhances DSB-induced HR by yKu-dependent and -independent mechanisms, with the latter mechanism promoting cell survival. Surprisingly, yku70 strains survived a DSB slightly better than wild type. We propose that this reflects enhanced HR, not by elimination of precise NHEJ since this pathway produces viable products, but by elimination of yKu-dependent interference of HR.
Collapse
Affiliation(s)
- J A Clikeman
- Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
| | | | | | | |
Collapse
|