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Paul T, Yan C, Yu J, Tsutakawa SE, Tainer JA, Wang D, Ivanov I. Molecular model of TFIIH recruitment to the transcription-coupled repair machinery. Nat Commun 2025; 16:2341. [PMID: 40057514 PMCID: PMC11890784 DOI: 10.1038/s41467-025-57593-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Accepted: 02/26/2025] [Indexed: 05/13/2025] Open
Abstract
Transcription-coupled repair (TCR) is a vital nucleotide excision repair sub-pathway that removes DNA lesions from actively transcribed DNA strands. Binding of CSB to lesion-stalled RNA Polymerase II (Pol II) initiates TCR by triggering the recruitment of downstream repair factors. Yet it remains unknown how transcription factor IIH (TFIIH) is recruited to the intact TCR complex. Combining existing structural data with AlphaFold predictions, we build an integrative model of the initial TFIIH-bound TCR complex. We show how TFIIH can be first recruited in an open repair-inhibited conformation, which requires subsequent CAK module removal and conformational closure to process damaged DNA. In our model, CSB, CSA, UVSSA, elongation factor 1 (ELOF1), and specific Pol II and UVSSA-bound ubiquitin moieties come together to provide interaction interfaces needed for TFIIH recruitment. STK19 acts as a linchpin of the assembly, orienting the incoming TFIIH and bridging Pol II to core TCR factors and DNA. Molecular simulations of the TCR-associated CRL4CSA ubiquitin ligase complex unveil the interplay of segmental DDB1 flexibility, continuous Cullin4A flexibility, and the key role of ELOF1 for Pol II ubiquitination that enables TCR. Collectively, these findings elucidate the coordinated assembly of repair proteins in early TCR.
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Affiliation(s)
- Tanmoy Paul
- Department of Chemistry, Georgia State University, Atlanta, GA, USA
- Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA
| | - Chunli Yan
- Department of Chemistry, Georgia State University, Atlanta, GA, USA
- Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA
| | - Jina Yu
- Department of Chemistry, Georgia State University, Atlanta, GA, USA
- Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA
| | - Susan E Tsutakawa
- Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
| | - John A Tainer
- Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
- Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Dong Wang
- Division of Pharmaceutical Sciences, Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, CA, San Diego, USA
| | - Ivaylo Ivanov
- Department of Chemistry, Georgia State University, Atlanta, GA, USA.
- Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA.
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Zachayus A, Loup-Forest J, Cura V, Poterszman A. Nucleotide Excision Repair: Insights into Canonical and Emerging Functions of the Transcription/DNA Repair Factor TFIIH. Genes (Basel) 2025; 16:231. [PMID: 40004560 PMCID: PMC11855273 DOI: 10.3390/genes16020231] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2025] [Revised: 01/31/2025] [Accepted: 02/06/2025] [Indexed: 02/27/2025] Open
Abstract
Nucleotide excision repair (NER) is a universal cut-and-paste DNA repair mechanism that corrects bulky DNA lesions such as those caused by UV radiation, environmental mutagens, and some chemotherapy drugs. In this review, we focus on the human transcription/DNA repair factor TFIIH, a key player of the NER pathway in eukaryotes. This 10-subunit multiprotein complex notably verifies the presence of a lesion and opens the DNA around the damage via its XPB and XPD subunits, two proteins identified in patients suffering from Xeroderma Pigmentosum syndrome. Isolated as a class II gene transcription factor in the late 1980s, TFIIH is a prototypic molecular machine that plays an essential role in both DNA repair and transcription initiation and harbors a DNA helicase, a DNA translocase, and kinase activity. More recently, TFIIH subunits have been identified as participating in other cellular processes, including chromosome segregation during mitosis, maintenance of mitochondrial DNA integrity, and telomere replication.
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Affiliation(s)
- Amélie Zachayus
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France; (A.Z.); (J.L.-F.); (V.C.)
- Centre National de la Recherche Scientifique (CNRS), UMR 7104, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Institut National De La Sante et de la Recherche Médicale (Inserm), UMR S 1258, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Equipe Labellisée Ligue Contre le Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
| | - Jules Loup-Forest
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France; (A.Z.); (J.L.-F.); (V.C.)
- Centre National de la Recherche Scientifique (CNRS), UMR 7104, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Institut National De La Sante et de la Recherche Médicale (Inserm), UMR S 1258, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Equipe Labellisée Ligue Contre le Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
| | - Vincent Cura
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France; (A.Z.); (J.L.-F.); (V.C.)
- Centre National de la Recherche Scientifique (CNRS), UMR 7104, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Institut National De La Sante et de la Recherche Médicale (Inserm), UMR S 1258, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Equipe Labellisée Ligue Contre le Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
| | - Arnaud Poterszman
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France; (A.Z.); (J.L.-F.); (V.C.)
- Centre National de la Recherche Scientifique (CNRS), UMR 7104, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Institut National De La Sante et de la Recherche Médicale (Inserm), UMR S 1258, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Equipe Labellisée Ligue Contre le Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
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Choi S, Shin M, Kim WY. Targeting the DNA damage response (DDR) of cancer cells with natural compounds derived from Panax ginseng and other plants. J Ginseng Res 2025; 49:1-11. [PMID: 39872282 PMCID: PMC11764321 DOI: 10.1016/j.jgr.2024.04.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2024] [Revised: 03/30/2024] [Accepted: 04/01/2024] [Indexed: 01/30/2025] Open
Abstract
DNA damage is a driver of cancer formation, leading to the impairment of repair mechanisms in cancer cells and rendering them susceptible to DNA-damaging therapeutic approaches. The concept of "synthetic lethality" in cancer clinics has emerged, particularly with the use of PARP inhibitors and the identification of DNA damage response (DDR) mutation biomarkers, emphasizing the significance of targeting DDR in cancer therapy. Novel approaches aimed at genome maintenance machinery are under development to further enhance the efficacy of cancer treatments. Natural compounds from traditional medicine, renowned for their anti-aging and anticarcinogenic properties, have garnered attention. Ginseng-derived compounds, in particular, exhibit anti-carcinogenic effects by suppressing reactive oxygen species (ROS) and protecting cells from DNA damage-induced carcinogenesis. However, the anticancer therapeutic effect of ginseng compounds has also been demonstrated by inducing DNA damage and blocking DDR. This review concentrates on the biphasic effects of ginseng compounds on DNA mutations-both inhibiting mutation accumulation and impairing DNA repair. Additionally, it explores other natural compounds targeting DDR directly, providing potential insights into enhancing cancer therapy efficacy.
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Affiliation(s)
- SeokGyeong Choi
- College of Pharmacy, Sookmyung Women's University, Seoul, Republic of Korea
| | - Minwook Shin
- College of Pharmacy, Sookmyung Women's University, Seoul, Republic of Korea
| | - Woo-Young Kim
- College of Pharmacy, Sookmyung Women's University, Seoul, Republic of Korea
- Muscle Physiome Research Center, Sookmyung Women's University, Seoul, Republic of Korea
- Research Institute of Pharmaceutical Sciences, Sookmyung Women's University, Seoul, Republic of Korea
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4
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Costanzo F, Paccosi E, Proietti-De-Santis L, Egly JM. CS proteins and ubiquitination: orchestrating DNA repair with transcription and cell division. Trends Cell Biol 2024; 34:882-895. [PMID: 38910038 DOI: 10.1016/j.tcb.2024.06.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Revised: 05/27/2024] [Accepted: 06/04/2024] [Indexed: 06/25/2024]
Abstract
To face genotoxic stress, eukaryotic cells evolved extremely refined mechanisms. Defects in counteracting the threat imposed by DNA damage underlie the rare disease Cockayne syndrome (CS), which arises from mutations in the CSA and CSB genes. Although initially defined as DNA repair proteins, recent work shows that CSA and CSB act instead as master regulators of the integrated response to genomic stress by coordinating DNA repair with transcription and cell division. CSA and CSB exert this function through the ubiquitination of target proteins, which are effectors/regulators of these processes. This review describes how the ubiquitination of target substrates is a common denominator by which CSA and CSB participate in different aspects of cellular life and how their mutation gives rise to the complex disease CS.
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Affiliation(s)
- Federico Costanzo
- Faculty of Biomedical Sciences, Institute of Oncology Research, USI, Bellinzona 6500, Switzerland; Department of Functional Genomics and Cancer, IGBMC, CNRS/INSERM/University of Strasbourg, Illkirch-Graffenstaden 67400, Strasbourg, France.
| | - Elena Paccosi
- Unit of Molecular Genetics of Aging, Department of Ecology and Biology, University of Tuscia, Viterbo 01100, Italy
| | - Luca Proietti-De-Santis
- Unit of Molecular Genetics of Aging, Department of Ecology and Biology, University of Tuscia, Viterbo 01100, Italy
| | - Jean Marc Egly
- Faculty of Biomedical Sciences, Institute of Oncology Research, USI, Bellinzona 6500, Switzerland; Department of Functional Genomics and Cancer, IGBMC, CNRS/INSERM/University of Strasbourg, Illkirch-Graffenstaden 67400, Strasbourg, France; College of Medicine, Centre for Genomics and Precision Medicine, National Taiwan University, Taipei City, Taiwan
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5
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D'Souza A, Kim M, Chazin WJ, Schärer OD. Protein-protein interactions in the core nucleotide excision repair pathway. DNA Repair (Amst) 2024; 141:103728. [PMID: 39029374 PMCID: PMC11330345 DOI: 10.1016/j.dnarep.2024.103728] [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: 05/15/2024] [Revised: 07/08/2024] [Accepted: 07/09/2024] [Indexed: 07/21/2024]
Abstract
Nucleotide excision repair (NER) clears genomes of DNA adducts formed by UV light, environmental agents, and antitumor drugs. Gene mutations that lead to defects in the core NER reaction cause the skin cancer-prone disease xeroderma pigmentosum. In NER, DNA lesions are excised within an oligonucleotide of 25-30 residues via a complex, multi-step reaction that is regulated by protein-protein interactions. These interactions were first characterized in the 1990s using pull-down, co-IP and yeast two-hybrid assays. More recently, high-resolution structures and detailed functional studies have started to yield detailed pictures of the progression along the NER reaction coordinate. In this review, we highlight how the study of interactions among proteins by structural and/or functional studies have provided insights into the mechanisms by which the NER machinery recognizes and excises DNA lesions. Furthermore, we identify reported, but poorly characterized or unsubstantiated interactions in need of further validation.
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Affiliation(s)
- Areetha D'Souza
- Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, the Republic of Korea; Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-7917, USA; Center for Structural Biology, Vandebilt University, Nashville, TN 37232-7917, USA
| | - Mihyun Kim
- Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, the Republic of Korea; Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, the Republic of Korea
| | - Walter J Chazin
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-7917, USA; Center for Structural Biology, Vandebilt University, Nashville, TN 37232-7917, USA; Department of Chemistry, Vanderbilt University, Nashville, TN 37232-7917, USA
| | - Orlando D Schärer
- Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, the Republic of Korea; Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, the Republic of Korea; Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-7917, USA.
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Yancoskie M, Khaleghi R, Gururajan A, Raghunathan A, Gupta A, Diethelm S, Maritz C, Sturla S, Krishnan M, Naegeli H. ASH1L guards cis-regulatory elements against cyclobutane pyrimidine dimer induction. Nucleic Acids Res 2024; 52:8254-8270. [PMID: 38884271 PMCID: PMC11317172 DOI: 10.1093/nar/gkae517] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Revised: 05/29/2024] [Accepted: 06/04/2024] [Indexed: 06/18/2024] Open
Abstract
The histone methyltransferase ASH1L, first discovered for its role in transcription, has been shown to accelerate the removal of ultraviolet (UV) light-induced cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair. Previous reports demonstrated that CPD excision is most efficient at transcriptional regulatory elements, including enhancers, relative to other genomic sites. Therefore, we analyzed DNA damage maps in ASH1L-proficient and ASH1L-deficient cells to understand how ASH1L controls enhancer stability. This comparison showed that ASH1L protects enhancer sequences against the induction of CPDs besides stimulating repair activity. ASH1L reduces CPD formation at C-containing but not at TT dinucleotides, and no protection occurs against pyrimidine-(6,4)-pyrimidone photoproducts or cisplatin crosslinks. The diminished CPD induction extends to gene promoters but excludes retrotransposons. This guardian role against CPDs in regulatory elements is associated with the presence of H3K4me3 and H3K27ac histone marks, which are known to interact with the PHD and BRD motifs of ASH1L, respectively. Molecular dynamics simulations identified a DNA-binding AT hook of ASH1L that alters the distance and dihedral angle between neighboring C nucleotides to disfavor dimerization. The loss of this protection results in a higher frequency of C->T transitions at enhancers of skin cancers carrying ASH1L mutations compared to ASH1L-intact counterparts.
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Affiliation(s)
- Michelle N Yancoskie
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich 8057, Switzerland
| | - Reihaneh Khaleghi
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich 8057, Switzerland
| | - Anirvinya Gururajan
- Center for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Hyderabad 500032, India
| | - Aadarsh Raghunathan
- Center for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Hyderabad 500032, India
| | - Aryan Gupta
- Center for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Hyderabad 500032, India
| | - Sarah Diethelm
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich 8057, Switzerland
| | - Corina Maritz
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich 8057, Switzerland
| | - Shana J Sturla
- Department of Health Sciences and Technology, ETH Zurich, Zurich 8092, Switzerland
| | - Marimuthu Krishnan
- Center for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Hyderabad 500032, India
| | - Hanspeter Naegeli
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich 8057, Switzerland
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Hoag A, Duan M, Mao P. The role of Transcription Factor IIH complex in nucleotide excision repair. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2024; 65 Suppl 1:72-81. [PMID: 37545038 PMCID: PMC10903506 DOI: 10.1002/em.22568] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/15/2023] [Revised: 07/05/2023] [Accepted: 08/03/2023] [Indexed: 08/08/2023]
Abstract
DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide excision repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers from ultraviolet light or DNA crosslinking damage from platinum-based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10-subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre-initiation complex and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG-NER) and transcription-coupled NER (TC-NER). We will also discuss how TFIIH's roles in the two subpathways lead to different genetic disorders.
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Affiliation(s)
- Allyson Hoag
- Department of Internal Medicine, University of New Mexico, Albuquerque, New Mexico, USA
- Comprehensive Cancer Center, University of New Mexico, Albuquerque, New Mexico, United States
| | - Mingrui Duan
- Department of Internal Medicine, University of New Mexico, Albuquerque, New Mexico, USA
- Comprehensive Cancer Center, University of New Mexico, Albuquerque, New Mexico, United States
| | - Peng Mao
- Department of Internal Medicine, University of New Mexico, Albuquerque, New Mexico, USA
- Comprehensive Cancer Center, University of New Mexico, Albuquerque, New Mexico, United States
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8
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Nouri N, Iravani P, Abtahi‐Naeini B. Sun protection behaviors among children aged 6-18 years old, the role of socioeconomic factors: A cross-sectional study. Health Sci Rep 2023; 6:e1727. [PMID: 38028698 PMCID: PMC10663172 DOI: 10.1002/hsr2.1727] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2023] [Revised: 10/18/2023] [Accepted: 11/05/2023] [Indexed: 12/01/2023] Open
Abstract
Background and Aims Evaluation of sun protection behavior and related factors in children aged between 6 and 18 years in Isfahan, Iran. Methods This cross-sectional study was conducted at outpatient pediatric clinics affiliated with Isfahan University of Medical Sciences, Isfahan, Iran during the summer of 2021. A checklist was designed, and the interviewer used that to gather the required information including children's demographic characteristics, families' socioeconomic data, and sun-protective behaviors in children. Results The mean ± standard deviation (SD) age of children was 10.54 ± 3.61. Among the study population, 63.7% of children were male. The most common skin phototypes were II and III (33.5% each). 83.8% of children used at least one method of sun protection (94.5% of girls, 77.6% of boys, p < 0.001). Wearing long-sleeved clothes was the most frequent UV-protection method (48%), while sunscreen application was the least prevalent method (28.1%). Sun protection behaviors were more frequent among urban children (86.7%, p = 0.009) and children with wealthy families (94%, p = 0.035). Sun protection methods were used by most of the children whose mothers applied sunscreen on a daily basis (90.2%, p = 0.002) and all of the children whose mothers held a master's degree or above (100%, p = 0.004). Conclusion Children's sun protection behavior is directly associated with demographic characteristics, families' socioeconomic level and maternal usage of sun protection measures. It is necessary to provide information and education about sun protection methods and the risks of excessive sun exposure to families and children, as well as facilitate their access to these.
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Affiliation(s)
- Nikta Nouri
- School of MedicineIsfahan University of Medical SciencesIsfahanIran
| | - Parisa Iravani
- Pediatrics Department, School of MedicineIsfahan University of Medical SciencesIsfahanIran
| | - Bahareh Abtahi‐Naeini
- Pediatric Dermatology Division of Pediatric Department, Imam Hossein Children's HospitalIsfahan University of Medical SciencesIsfahanIran
- Skin Diseases and Leishmaniasis Research CenterIsfahan University of Medical SciencesIsfahanIran
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Maritz C, Khaleghi R, Yancoskie MN, Diethelm S, Brülisauer S, Ferreira NS, Jiang Y, Sturla SJ, Naegeli H. ASH1L-MRG15 methyltransferase deposits H3K4me3 and FACT for damage verification in nucleotide excision repair. Nat Commun 2023; 14:3892. [PMID: 37393406 PMCID: PMC10314917 DOI: 10.1038/s41467-023-39635-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2022] [Accepted: 06/22/2023] [Indexed: 07/03/2023] Open
Abstract
To recognize DNA adducts, nucleotide excision repair (NER) deploys the XPC sensor, which detects damage-induced helical distortions, followed by engagement of TFIIH for lesion verification. Accessory players ensure that this factor handover takes place in chromatin where DNA is tightly wrapped around histones. Here, we describe how the histone methyltransferase ASH1L, once activated by MRG15, helps XPC and TFIIH to navigate through chromatin and induce global-genome NER hotspots. Upon UV irradiation, ASH1L adds H3K4me3 all over the genome (except in active gene promoters), thus priming chromatin for XPC relocations from native to damaged DNA. The ASH1L-MRG15 complex further recruits the histone chaperone FACT to DNA lesions. In the absence of ASH1L, MRG15 or FACT, XPC is misplaced and persists on damaged DNA without being able to deliver the lesions to TFIIH. We conclude that ASH1L-MRG15 makes damage verifiable by the NER machinery through the sequential deposition of H3K4me3 and FACT.
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Affiliation(s)
- Corina Maritz
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Reihaneh Khaleghi
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Michelle N Yancoskie
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Sarah Diethelm
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Sonja Brülisauer
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Natalia Santos Ferreira
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Yang Jiang
- Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland
| | - Shana J Sturla
- Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland
| | - Hanspeter Naegeli
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland.
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10
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Yu J, Yan C, Dodd T, Tsai CL, Tainer JA, Tsutakawa SE, Ivanov I. Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases. Nat Commun 2023; 14:2758. [PMID: 37179334 PMCID: PMC10183003 DOI: 10.1038/s41467-023-38416-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2022] [Accepted: 04/28/2023] [Indexed: 05/15/2023] Open
Abstract
Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH's global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.
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Affiliation(s)
- Jina Yu
- Department of Chemistry, Georgia State University, Atlanta, GA, USA
- Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA
| | - Chunli Yan
- Department of Chemistry, Georgia State University, Atlanta, GA, USA
- Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA
| | - Thomas Dodd
- Department of Chemistry, Georgia State University, Atlanta, GA, USA
- Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA
| | - Chi-Lin Tsai
- Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - John A Tainer
- Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
| | - Susan E Tsutakawa
- Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
| | - Ivaylo Ivanov
- Department of Chemistry, Georgia State University, Atlanta, GA, USA.
- Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA.
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11
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Yancoskie MN, Maritz C, van Eijk P, Reed SH, Naegeli H. To incise or not and where: SET-domain methyltransferases know. Trends Biochem Sci 2023; 48:321-330. [PMID: 36357311 DOI: 10.1016/j.tibs.2022.10.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2022] [Revised: 10/10/2022] [Accepted: 10/14/2022] [Indexed: 11/09/2022]
Abstract
The concept of the histone code posits that histone modifications regulate gene functions once interpreted by epigenetic readers. A well-studied case is trimethylation of lysine 4 of histone H3 (H3K4me3), which is enriched at gene promoters. However, H3K4me3 marks are not needed for the expression of most genes, suggesting extra roles, such as influencing the 3D genome architecture. Here, we highlight an intriguing analogy between the H3K4me3-dependent induction of double-strand breaks in several recombination events and the impact of this same mark on DNA incisions for the repair of bulky lesions. We propose that Su(var)3-9, Enhancer-of-zeste and Trithorax (SET)-domain methyltransferases generate H3K4me3 to guide nucleases into chromatin spaces, the favorable accessibility of which ensures that DNA break intermediates are readily processed, thereby safeguarding genome stability.
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Affiliation(s)
- Michelle N Yancoskie
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Corina Maritz
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Patrick van Eijk
- Institute of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, UK
| | - Simon H Reed
- Institute of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, UK
| | - Hanspeter Naegeli
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland.
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12
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Bralić A, Tehseen M, Sobhy MA, Tsai CL, Alhudhali L, Yi G, Yu J, Yan C, Ivanov I, Tsutakawa SE, Tainer J, Hamdan S. A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair. Nucleic Acids Res 2022; 51:1019-1033. [PMID: 36477609 PMCID: PMC9943652 DOI: 10.1093/nar/gkac1095] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 10/21/2022] [Accepted: 11/26/2022] [Indexed: 12/12/2022] Open
Abstract
Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.
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Affiliation(s)
- Amer Bralić
- Bioscience Program, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
| | - Muhammad Tehseen
- Bioscience Program, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
| | - Mohamed A Sobhy
- Bioscience Program, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
| | - Chi-Lin Tsai
- Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Lubna Alhudhali
- Bioscience Program, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
| | - Gang Yi
- Bioscience Program, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
| | - Jina Yu
- Department of Chemistry, Georgia State University, Atlanta, GA 30302 USA; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, 30302, USA
| | - Chunli Yan
- Department of Chemistry, Georgia State University, Atlanta, GA 30302 USA; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, 30302, USA
| | - Ivaylo Ivanov
- Department of Chemistry, Georgia State University, Atlanta, GA 30302 USA; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, 30302, USA
| | - Susan E Tsutakawa
- Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
| | - John A Tainer
- Correspondence may also be addressed to John A. Tainer. Tel: +1 713 563 7725; Fax: +1 713 794 3270;
| | - Samir M Hamdan
- To whom correspondence should be addressed. Tel: +96 628082384; Cell: +96 6544700031;
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13
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Kim M, Kim HS, D’Souza A, Gallagher K, Jeong E, Topolska-Woś A, Ogorodnik Le Meur K, Tsai CL, Tsai MS, Kee M, Tainer JA, Yeo JE, Chazin WJ, Schärer OD. Two interaction surfaces between XPA and RPA organize the preincision complex in nucleotide excision repair. Proc Natl Acad Sci U S A 2022; 119:e2207408119. [PMID: 35969784 PMCID: PMC9407234 DOI: 10.1073/pnas.2207408119] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Accepted: 07/04/2022] [Indexed: 12/15/2022] Open
Abstract
The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.
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Affiliation(s)
- Mihyun Kim
- Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
| | - Hyun-Suk Kim
- Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, Republic of Korea
| | - Areetha D’Souza
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-7917
- Center for Structural Biology, Vanderbilt University, Nashville, TN 37232-7917
| | - Kaitlyn Gallagher
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-7917
- Center for Structural Biology, Vanderbilt University, Nashville, TN 37232-7917
| | - Eunwoo Jeong
- Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, Republic of Korea
| | - Agnieszka Topolska-Woś
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-7917
- Center for Structural Biology, Vanderbilt University, Nashville, TN 37232-7917
| | - Kateryna Ogorodnik Le Meur
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-7917
- Center for Structural Biology, Vanderbilt University, Nashville, TN 37232-7917
| | - Chi-Lin Tsai
- Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030
| | - Miaw-Sheue Tsai
- Biological and Systems Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
| | - Minyong Kee
- Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, Republic of Korea
| | - John A. Tainer
- Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030
| | - Jung-Eun Yeo
- Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, Republic of Korea
| | - Walter J. Chazin
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-7917
- Center for Structural Biology, Vanderbilt University, Nashville, TN 37232-7917
- Department of Chemistry, Vanderbilt University, Nashville, TN 37232-7917
| | - Orlando D. Schärer
- Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-7917
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14
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Transcription associated cyclin-dependent kinases as therapeutic targets for prostate cancer. Oncogene 2022; 41:3303-3315. [PMID: 35568739 PMCID: PMC9187515 DOI: 10.1038/s41388-022-02347-1] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2022] [Revised: 04/21/2022] [Accepted: 05/04/2022] [Indexed: 11/08/2022]
Abstract
Transcriptional deregulation has emerged as a hallmark of several cancer types. In metastatic castration-resistant prostate cancer, a stage in which systemic androgen deprivation therapies fail to show clinical benefit, transcriptional addiction to the androgen receptor is maintained in most patients. This has led to increased efforts to find novel therapies that prevent oncogenic transactivation of the androgen receptor. In this context, a group of druggable protein kinases, known as transcription associated cyclin-dependent kinases (tCDKs), show great potential as therapeutic targets. Despite initial reservations about targeting tCDKs due to their ubiquitous and prerequisite nature, preclinical studies showed that selectively inhibiting such kinases could provide sufficient therapeutic window to exert antitumour effects in the absence of systemic toxicity. As a result, several highly specific inhibitors are currently being trialled in solid tumours, including prostate cancer. This article summarises the roles of tCDKs in regulating gene transcription and highlights rationales for their targeting in prostate cancer. It provides an overview of the most recent developments in this therapeutic area, including the most recent clinical advances, and discusses the utility of tCDK inhibitors in combination with established cancer agents.
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15
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Templeton CW, Traktman P. UV Irradiation of Vaccinia Virus-Infected Cells Impairs Cellular Functions, Introduces Lesions into the Viral Genome, and Uncovers Repair Capabilities for the Viral Replication Machinery. J Virol 2022; 96:e0213721. [PMID: 35404095 PMCID: PMC9093118 DOI: 10.1128/jvi.02137-21] [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: 12/15/2021] [Accepted: 03/17/2022] [Indexed: 11/20/2022] Open
Abstract
Vaccinia virus (VV), the prototypic poxvirus, encodes a repertoire of proteins responsible for the metabolism of its large dsDNA genome. Previous work has furthered our understanding of how poxviruses replicate and recombine their genomes, but little is known about whether the poxvirus genome undergoes DNA repair. Our studies here are aimed at understanding how VV responds to exogenous DNA damage introduced by UV irradiation. Irradiation of cells prior to infection decreased protein synthesis and led to an ∼12-fold reduction in viral yield. On top of these cell-specific insults, irradiation of VV infections at 4 h postinfection (hpi) introduced both cyclobutene pyrimidine dimer (CPD) and 6,4-photoproduct (6,4-PP) lesions into the viral genome led to a nearly complete halt to further DNA synthesis and to a further reduction in viral yield (∼35-fold). DNA lesions persisted throughout infection and were indeed present in the genomes encapsidated into nascent virions. Depletion of several cellular proteins that mediate nucleotide excision repair (XP-A, -F, and -G) did not render viral infections hypersensitive to UV. We next investigated whether viral proteins were involved in combatting DNA damage. Infections performed with a virus lacking the A50 DNA ligase were moderately hypersensitive to UV irradiation (∼3-fold). More strikingly, when the DNA polymerase inhibitor cytosine arabinoside (araC) was added to wild-type infections at the time of UV irradiation (4 hpi), an even greater hypersensitivity to UV irradiation was seen (∼11-fold). Virions produced under the latter condition contained elevated levels of CPD adducts, strongly suggesting that the viral polymerase contributes to the repair of UV lesions introduced into the viral genome. IMPORTANCE Poxviruses remain of significant interest because of their continuing clinical relevance, their utility for the development of vaccines and oncolytic therapies, and their illustration of fundamental principles of viral replication and virus/cell interactions. These viruses are unique in that they replicate exclusively in the cytoplasm of infected mammalian cells, providing novel challenges for DNA viruses. How poxviruses replicate, recombine, and possibly repair their genomes is still only partially understood. Using UV irradiation as a form of exogenous DNA damage, we have examined how vaccinia virus metabolizes its genome following insult. We show that even UV irradiation of cells prior to infection diminishes viral yield, while UV irradiation during infection damages the genome, causes a halt in DNA accumulation, and reduces the viral yield more severely. Furthermore, we show that viral proteins, but not the cellular machinery, contribute to a partial repair of the viral genome following UV irradiation.
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Affiliation(s)
- Conor W. Templeton
- Departments of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Paula Traktman
- Departments of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA
- Departments of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA
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16
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D'Souza A, Blee AM, Chazin WJ. Mechanism of action of nucleotide excision repair machinery. Biochem Soc Trans 2022; 50:375-386. [PMID: 35076656 PMCID: PMC9275815 DOI: 10.1042/bst20210246] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2021] [Revised: 12/20/2021] [Accepted: 12/22/2021] [Indexed: 11/08/2023]
Abstract
Nucleotide excision repair (NER) is a versatile DNA repair pathway essential for the removal of a broad spectrum of structurally diverse DNA lesions arising from a variety of sources, including UV irradiation and environmental toxins. Although the core factors and basic stages involved in NER have been identified, the mechanisms of the NER machinery are not well understood. This review summarizes our current understanding of the mechanisms and order of assembly in the core global genome (GG-NER) pathway.
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Affiliation(s)
- Areetha D'Souza
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37240-7917, U.S.A
| | - Alexandra M Blee
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37240-7917, U.S.A
| | - Walter J Chazin
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37240-7917, U.S.A
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17
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Chauhan AK, Sun Y, Zhu Q, Wani AA. Timely upstream events regulating nucleotide excision repair by ubiquitin-proteasome system: ubiquitin guides the way. DNA Repair (Amst) 2021; 103:103128. [PMID: 33991872 DOI: 10.1016/j.dnarep.2021.103128] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 04/15/2021] [Accepted: 05/04/2021] [Indexed: 12/11/2022]
Abstract
The ubiquitin-proteasome system (UPS) plays crucial roles in regulation of multiple DNA repair pathways, including nucleotide excision repair (NER), which eliminates a broad variety of helix-distorting DNA lesions that can otherwise cause deleterious mutations and genomic instability. In mammalian NER, DNA damage sensors, DDB and XPC acting in global genomic NER (GG-NER), and, CSB and RNAPII acting in transcription-coupled NER (TC-NER) sub-pathways, undergo an array of post-translational ubiquitination at the DNA lesion sites. Accumulating evidence indicates that ubiquitination orchestrates the productive assembly of NER preincision complex by driving well-timed compositional changes in DNA damage-assembled sensor complexes. Conversely, the deubiquitination is also intimately involved in regulating the damage sensing aftermath, via removal of degradative ubiquitin modification on XPC and CSB to prevent their proteolysis for the factor recycling. This review summaries the relevant research efforts and latest findings in our understanding of ubiquitin-mediated regulation of NER and active participation by new regulators of NER, e.g., Cullin-Ring ubiquitin ligases (CRLs), ubiquitin-specific proteases (USPs) and ubiquitin-dependent segregase, valosin-containing protein (VCP)/p97. We project hypothetical step-by-step models in which VCP/p97-mediated timely extraction of damage sensors is integral to overall productive NER. The USPs and proteasome subtly counteract in fine-tuning the vital stability and function of NER damage sensors.
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Affiliation(s)
- Anil K Chauhan
- Department of Radiology, The Ohio State University, Columbus, OH, 43210, United States
| | - Yingming Sun
- Department of Radiology, The Ohio State University, Columbus, OH, 43210, United States
| | - Qianzheng Zhu
- Department of Radiology, The Ohio State University, Columbus, OH, 43210, United States.
| | - Altaf A Wani
- Department of Radiology, The Ohio State University, Columbus, OH, 43210, United States; Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH, 43210, United States; James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH, 43210, United States.
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18
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Latimer JJ, Alhamed A, Sveiven S, Almutairy A, Klimas NG, Abreu M, Sullivan K, Grant SG. Preliminary Evidence for a Hormetic Effect on DNA Nucleotide Excision Repair in Veterans with Gulf War Illness. Mil Med 2021; 185:e47-e52. [PMID: 31334811 PMCID: PMC7353836 DOI: 10.1093/milmed/usz177] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2019] [Revised: 06/17/2019] [Accepted: 06/25/2019] [Indexed: 12/13/2022] Open
Abstract
Introduction Veterans of the 1991 Gulf War were potentially exposed to a mixture of stress, chemicals and radiation that may have contributed to the persistent symptoms of Gulf War Illness (GWI). The genotoxic effects of some of these exposures are mediated by the DNA nucleotide excision repair (NER) pathway. We hypothesized that individuals with relatively low DNA repair capacity would suffer greater damage from cumulative genotoxic exposures, some of which would persist, causing ongoing problems. Materials and Methods Blood samples were obtained from symptomatic Gulf War veterans and age-matched controls. The unscheduled DNA synthesis assay, a functional measurement of NER capacity, was performed on cultured lymphocytes, and lymphocyte mRNA was extracted and analyzed by sequencing. Results Despite our hypothesis that GWI would be associated with DNA repair deficiency, NER capacity in lymphocytes from affected GWI veterans actually exhibited a significantly elevated level of DNA repair (p = 0.016). Both total gene expression and NER gene expression successfully differentiated individuals with GWI from unaffected controls. The observed functional increase in DNA repair capacity was accompanied by an overexpression of genes in the NER pathway, as determined by RNA sequencing analysis. Conclusion We suggest that the observed elevations in DNA repair capacity and NER gene expression are indicative of a “hormetic,” i.e., induced or adaptive protective response to battlefield exposures. Normally such effects are short-term, but in these individuals this response has resulted in a long-term metabolic shift that may also be responsible for the persistent symptoms of GWI.
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Affiliation(s)
- Jean J Latimer
- Department of Pharmaceutical Sciences, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328.,South University Drive, AutoNation Institute for Breast Cancer Research, 3321, Fort Lauderdale, FL 33328
| | - Abdullah Alhamed
- Department of Pharmaceutical Sciences, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328.,South University Drive, AutoNation Institute for Breast Cancer Research, 3321, Fort Lauderdale, FL 33328
| | - Stefanie Sveiven
- South University Drive, AutoNation Institute for Breast Cancer Research, 3321, Fort Lauderdale, FL 33328
| | - Ali Almutairy
- Department of Pharmaceutical Sciences, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328.,South University Drive, AutoNation Institute for Breast Cancer Research, 3321, Fort Lauderdale, FL 33328
| | - Nancy G Klimas
- Department of Clinical Immunology, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328.,Department of Medicine, Miami VA Healthcare System, 1201 NW 16th St, Miami, FL 33313
| | - Maria Abreu
- Department of Clinical Immunology, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328
| | - Kimberly Sullivan
- Department of Environmental Health, Boston University School of Public Health, 715 Albany St, Boston, MA 02118
| | - Stephen G Grant
- South University Drive, AutoNation Institute for Breast Cancer Research, 3321, Fort Lauderdale, FL 33328.,Department of Public Health, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328
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Abstract
Cyclin-dependent kinase 7 (CDK7), along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs progression through the cell cycle via T-loop phosphorylation of cell cycle CDKs. CAK is also a component of the general transcription factor, TFIIH. CDK7-mediated phosphorylation of RNA polymerase II (Pol II) at active gene promoters permits transcription. Cell cycle dysregulation is an established hallmark of cancer, and aberrant control of transcriptional processes, through diverse mechanisms, is also common in many cancers. Furthermore, CDK7 levels are elevated in a number of cancer types and are associated with clinical outcomes, suggestive of greater dependence on CDK7 activity, compared with normal tissues. These findings identify CDK7 as a cancer therapeutic target, and several recent publications report selective CDK7 inhibitors (CDK7i) with activity against diverse cancer types. Preclinical studies have shown that CDK7i cause cell cycle arrest, apoptosis and repression of transcription, particularly of super-enhancer-associated genes in cancer, and have demonstrated their potential for overcoming resistance to cancer treatments. Moreover, combinations of CDK7i with other targeted cancer therapies, including BET inhibitors, BCL2 inhibitors and hormone therapies, have shown efficacy in model systems. Four CDK7i, ICEC0942 (CT7001), SY-1365, SY-5609 and LY3405105, have now progressed to Phase I/II clinical trials. Here we describe the work that has led to the development of selective CDK7i, the current status of the most advanced clinical candidates, and discuss their potential importance as cancer therapeutics, both as monotherapies and in combination settings. ClinicalTrials.gov Identifiers: NCT03363893; NCT03134638; NCT04247126; NCT03770494.
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20
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Chauhan AK, Li P, Sun Y, Wani G, Zhu Q, Wani AA. Spironolactone-induced XPB degradation requires TFIIH integrity and ubiquitin-selective segregase VCP/p97. Cell Cycle 2020; 20:81-95. [PMID: 33381997 PMCID: PMC7849777 DOI: 10.1080/15384101.2020.1860559] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Mineralocorticoid and androgen receptor antagonist, spironolactone, was recently identified as an inhibitor of nucleotide excision repair (NER), acting via induction of proteolysis of TFIIH component Xeroderma Pigmentosum B protein (XPB). This activity provides a strong rationale for repurposing spironolactone for cancer therapy. Here, we report that the spironolactone-induced XPB proteolysis is mediated through ubiquitin-selective segregase, valosin-containing protein (VCP)/p97. We show that spironolactone induces a dose- and time-dependent degradation of XPB but not XPD, and that the XPB degradation is blocked by VCP/p97 inhibitors DBeQ, NMS-873, and neddylation inhibitor MLN4924. Moreover, the cellular treatment by VCP/p97 inhibitors leads to the accumulation of ubiquitin conjugates of XPB but not XPD. VCP/p97 knockdown by inducible shRNA does not affect XPB level but compromises the spironolactone-induced XPB degradation. Also, VCP/p97 interacts with XPB upon treatment of spironolactone and proteasome inhibitor MG132, while the VCP/p97 adaptor UBXD7 binds XPB and its ubiquitin conjugates. Additionally, ATP analog-mediated inhibition of Cdk7 significantly decelerates spironolactone-induced XPB degradation. Likewise, engaging TFIIH to NER by UV irradiation slows down spironolactone-induced XPB degradation. These results indicate that the spironolactone-induced XPB proteolysis requires VCP/p97 function and that XPB within holo-TFIIH rather than core-TFIIH is more vulnerable to spironolactone-induced proteolysis. Abbreviations
NER: nucleotide excision repair; TFIIH: transcription factor II H; CAK: Cdk-activating kinase (CAK) complex; XPB: Xeroderma Pigmentosum type B; VCP/p97: valosin-containing protein/p97; Cdk7: cyclin-dependent kinase 7; NAE: NEDD8-activating enzyme; IP: immunoprecipitation
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Affiliation(s)
- Anil K Chauhan
- Department of Radiology, The Ohio State University , Columbus, OH, USA
| | - Ping Li
- Department of Radiology, The Ohio State University , Columbus, OH, USA
| | - Yingming Sun
- Department of Radiology, The Ohio State University , Columbus, OH, USA
| | - Gulzar Wani
- Department of Radiology, The Ohio State University , Columbus, OH, USA
| | - Qianzheng Zhu
- Department of Radiology, The Ohio State University , Columbus, OH, USA
| | - Altaf A Wani
- Department of Radiology, The Ohio State University , Columbus, OH, USA.,Department of Molecular and Cellular Biochemistry, The Ohio State University , Columbus, OH, USA.,James Cancer Hospital and Solove Research Institute, The Ohio State University , Columbus, OH, USA
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21
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Kathuria P, Singh P, Sharma P, Manderville RA, Wetmore SD. Molecular Dynamics Study of One-Base Deletion Duplexes Containing the Major DNA Adduct Formed by Ochratoxin A: Effects of Sequence Context and Adduct Ionization State on Lesion Site Structure and Mutagenicity. J Phys Chem B 2019; 123:6980-6989. [PMID: 31311268 DOI: 10.1021/acs.jpcb.9b06489] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Ochratoxin A (OTA) is a ubiquitous food toxin associated with chronic nephropathy in humans and renal carcinogenicity in rodents. The mutational spectra of cells exposed to OTA reveal that one-base deletions comprise the largest percentage (73%) of the total mutations that occur upon OTA exposure. To contribute toward understanding the prevalence of OTA-induced one-base deletion mutations, the present work uses molecular dynamics (MD) simulations to analyze the conformational preferences of one-base deletion duplexes containing OT-G, the major OTA adduct (addition product) at the C8-site of guanine. Specifically, the influence of OT-G in four possible ionization states and three sequence contexts (G1, G2 and G3 in the NarI (5'-G1G2CG3CC-3'), a prokaryotic mutational hotspot sequence) on the structure of the adducted DNA is investigated. Our data reveal that the damaged helices are stable in two (B-type (B) and stacked (S)) conformations that are structurally similar to those adopted by common N-linked C8-guanine lesions. However, the adduct ionization state and sequence context affect the degree of helical distortion and the B/S conformational heterogeneity, which will impact the lesion repair and replication outcomes. This finding correlates with the experimentally reported tissue-specific mutagenicity of OTA exposure. Furthermore, regardless of the adduct conformation, ionization state, or sequence context, more stable lesion-site interactions and lack of disruption of the flanking base pairs in the one-base deletion duplexes compared to the corresponding two-base deletion helices rationalize the greater abundance of OTA induced one-base deletions. Overall, our work provides valuable structural insights that help explain the experimentally observed mutagenicity associated with OTA.
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Affiliation(s)
- Preetleen Kathuria
- Computational Biochemistry Laboratory, Department of Chemistry and Centre for Advanced Studies in Chemistry , Panjab University , Chandigarh 160014 , India
| | - Prebhleen Singh
- Computational Biochemistry Laboratory, Department of Chemistry and Centre for Advanced Studies in Chemistry , Panjab University , Chandigarh 160014 , India
| | - Purshotam Sharma
- Computational Biochemistry Laboratory, Department of Chemistry and Centre for Advanced Studies in Chemistry , Panjab University , Chandigarh 160014 , India
| | - Richard A Manderville
- Departments of Chemistry and Toxicology , University of Guelph , Guelph , Ontario N1G 2W1 , Canada
| | - Stacey D Wetmore
- Department of Chemistry and Biochemistry , University of Lethbridge , Lethbridge , Alberta T1K 3M4 , Canada
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22
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Polynuclear ruthenium organometallic compounds induce DNA damage in human cells identified by the nucleotide excision repair factor XPC. Biosci Rep 2019; 39:BSR20190378. [PMID: 31227614 PMCID: PMC6629949 DOI: 10.1042/bsr20190378] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2019] [Revised: 06/14/2019] [Accepted: 06/19/2019] [Indexed: 12/13/2022] Open
Abstract
Ruthenium organometallic compounds represent an attractive avenue in developing alternatives to platinum-based chemotherapeutic agents. While evidence has been presented indicating ruthenium-based compounds interact with isolated DNA in vitro, it is unclear what effect these compounds exert in cells. Moreover, the antibiotic efficacy of polynuclear ruthenium organometallic compounds remains uncertain. In the present study, we report that exposure to polynuclear ruthenium organometallic compounds induces recruitment of damaged DNA sensing protein Xeroderma pigmentosum Group C into chromatin-immobilized foci. Additionally, we observed one of the tested polynuclear ruthenium organometallic compounds displayed increased cytotoxicity against human cells deficient in nucleotide excision repair (NER). Taken together, these results suggest that polynuclear ruthenium organometallic compounds induce DNA damage in cells, and that cellular resistance to these compounds may be influenced by the NER DNA repair phenotype of the cells.
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23
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Abstract
The nucleotide excision repair (NER) system removes a variety of types of helix-distorting lesions from DNA through a dual incision mechanism, in which the damaged nucleotide bases are excised in the form of a small, excised, damage-containing single-stranded DNA oligonucleotide (sedDNA). Damage removal leaves a gap in the DNA template that must then be filled in by the action of a DNA polymerase and ligated to the downstream phosphodiester backbone in the DNA to complete the repair reaction. Defects in damage removal, sedDNA processing, or gap filling have the potential to be mutagenic and lethal to cells, and thus several human pathologies, including cancer and aging, are associated with defects in NER. This review summarizes our current understanding of NER with a focus on the enzymes that excise sedDNAs and restore the duplex DNA to its native state in human cells.
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Affiliation(s)
- Michael G Kemp
- Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, OH, United States.
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24
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TFIIE orchestrates the recruitment of the TFIIH kinase module at promoter before release during transcription. Nat Commun 2019; 10:2084. [PMID: 31064989 PMCID: PMC6504876 DOI: 10.1038/s41467-019-10131-1] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2018] [Accepted: 04/18/2019] [Indexed: 11/08/2022] Open
Abstract
In eukaryotes, the general transcription factors TFIIE and TFIIH assemble at the transcription start site with RNA Polymerase II. However, the mechanism by which these transcription factors incorporate the preinitiation complex and coordinate their action during RNA polymerase II transcription remains elusive. Here we show that the TFIIEα and TFIIEβ subunits anchor the TFIIH kinase module (CAK) within the preinitiation complex. In addition, we show that while RNA polymerase II phosphorylation and DNA opening occur, CAK and TFIIEα are released from the promoter. This dissociation is impeded by either ATP-γS or CDK7 inhibitor THZ1, but still occurs when XPB activity is abrogated. Finally, we show that the Core-TFIIH and TFIIEβ are subsequently removed, while elongation factors such as DSIF are recruited. Remarkably, these early transcriptional events are affected by TFIIE and TFIIH mutations associated with the developmental disorder, trichothiodystrophy. The general transcription factors TFIIE and TFIIH assemble at the transcription start site with RNA Polymerase II. Here the authors provide evidence that the TFIIEα and TFIIEβ subunits anchor the TFIIH kinase module within the preinitiation complex before their release during transcription.
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25
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Wood DJ, Endicott JA. Structural insights into the functional diversity of the CDK-cyclin family. Open Biol 2019; 8:rsob.180112. [PMID: 30185601 PMCID: PMC6170502 DOI: 10.1098/rsob.180112] [Citation(s) in RCA: 168] [Impact Index Per Article: 28.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2018] [Accepted: 08/10/2018] [Indexed: 12/17/2022] Open
Abstract
Since their characterization as conserved modules that regulate progression through the eukaryotic cell cycle, cyclin-dependent protein kinases (CDKs) in higher eukaryotic cells are now also emerging as significant regulators of transcription, metabolism and cell differentiation. The cyclins, though originally characterized as CDK partners, also have CDK-independent roles that include the regulation of DNA damage repair and transcriptional programmes that direct cell differentiation, apoptosis and metabolic flux. This review compares the structures of the members of the CDK and cyclin families determined by X-ray crystallography, and considers what mechanistic insights they provide to guide functional studies and distinguish CDK- and cyclin-specific activities. Aberrant CDK activity is a hallmark of a number of diseases, and structural studies can provide important insights to identify novel routes to therapy.
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Affiliation(s)
- Daniel J Wood
- Newcastle Cancer Centre, Northern Institute for Cancer Research, Medical School, Newcastle University, Paul O'Gorman Building, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
| | - Jane A Endicott
- Newcastle Cancer Centre, Northern Institute for Cancer Research, Medical School, Newcastle University, Paul O'Gorman Building, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
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26
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Ueda M, Matsuura K, Kawai H, Wakasugi M, Matsunaga T. Spironolactone-induced XPB degradation depends on CDK7 kinase and SCF FBXL18 E3 ligase. Genes Cells 2019; 24:284-296. [PMID: 30762924 DOI: 10.1111/gtc.12674] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2018] [Revised: 01/30/2019] [Accepted: 02/09/2019] [Indexed: 12/27/2022]
Abstract
The multisubunit complex transcription factor IIH (TFIIH) has dual functions in transcriptional initiation and nucleotide excision repair (NER). TFIIH is comprised of two subcomplexes, the core subcomplex (seven subunits) including XPB and XPD helicases and the cyclin-dependent kinase (CDK)-activating kinase (CAK) subcomplex (three subunits) containing CDK7 kinase. Recently, it has been reported that spironolactone, an anti-aldosterone drug, inhibits cellular NER by inducing proteasomal degradation of XPB and potentiates the cytotoxicity of platinum-based drugs in cancer cells, suggesting possible drug repositioning. In this study, we have tried to uncover the mechanism underlying the chemical-induced XPB destabilization. Based on siRNA library screening and subsequent analyses, we identified SCFFBXL18 E3 ligase consisting of Skp1, Cul1, F-box protein FBXL18 and Rbx1 responsible for spironolactone-induced XPB polyubiquitination and degradation. In addition, we showed that CDK7 kinase activity is required for this process. Finally, we found that the Ser90 residue of XPB is essential for the chemical-induced destabilization. These results led us to propose a model that spironolactone may trigger the phosphorylation of XPB at Ser90 by CDK7, which promotes the recognition and polyubiquitination of XPB by SCFFBXL18 for proteasomal degradation.
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Affiliation(s)
- Masanobu Ueda
- Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Kenkyo Matsuura
- Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Hidehiko Kawai
- Department of Experimental Oncology, Research Center for Radiation Genome Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Mitsuo Wakasugi
- Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Tsukasa Matsunaga
- Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
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27
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Sugasawa K. Mechanism and regulation of DNA damage recognition in mammalian nucleotide excision repair. DNA Repair (Amst) 2019; 45:99-138. [DOI: 10.1016/bs.enz.2019.06.004] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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28
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Mullenders LHF. Solar UV damage to cellular DNA: from mechanisms to biological effects. Photochem Photobiol Sci 2018; 17:1842-1852. [PMID: 30065996 DOI: 10.1039/c8pp00182k] [Citation(s) in RCA: 135] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Solar ultraviolet (UV) radiation generates bulky photodimers at di-pyrimidine sites that pose stress to cells and organisms by hindering DNA replication and transcription. In addition, solar UV also induces various types of oxidative DNA lesions and single strand DNA breaks. Relieving toxicity and maintenance of genomic integrity are of clinical importance in relation to erythema/edema and diseases such as cancer, neurodegeneration and premature ageing, respectively. Following solar UV radiation, a network of DNA damage response mechanisms triggers a signal transduction cascade to regulate various genome-protection pathways including DNA damage repair, cell cycle control, apoptosis, transcription and chromatin remodeling. The effects of UVC and UVB radiation on cellular DNA are predominantly accounted for by the formation of photodimers at di-pyrimidine sites. These photodimers are mutagenic: UVC, UVB and also UVA radiation induce a broadly similar pattern of transition mutations at di-pyrimidine sites. The mutagenic potency of solar UV is counteracted by efficient repair of photodimers involving global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER); the latter is a specialized repair pathway to remove transcription-blocking photodimers and restore UV-inhibited transcription. On the molecular level these processes are facilitated and regulated by various post-translational modifications of NER factors and the chromatin substrate. Inherited defects in NER are manifested in different diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), UV sensitive syndrome (UVsS) and the photosensitive form of trichothiodystrophy (TTD). XP patients are prone to sunlight-induced skin cancer. UVB irradiated XP and CS knockout mouse models unveiled that only TC-NER counteracts erythema/edema, whereas both GG-NER and TC-NER protect against UVB-induced cancer. Additionally, UVA radiation induces mutations characterized by oxidation-linked signature at non-di-pyrimidine sites. The biological relevance of oxidation damage is demonstrated by the cancer susceptibility of UVB-irradiated mice deficient in repair of oxidation damage, i.e., 8-oxoguanine.
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29
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Mu H, Geacintov NE, Broyde S, Yeo JE, Schärer OD. Molecular basis for damage recognition and verification by XPC-RAD23B and TFIIH in nucleotide excision repair. DNA Repair (Amst) 2018; 71:33-42. [PMID: 30174301 DOI: 10.1016/j.dnarep.2018.08.005] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Global genome nucleotide excision repair (GG-NER) is the main pathway for the removal of bulky lesions from DNA and is characterized by an extraordinarily wide substrate specificity. Remarkably, the efficiency of lesion removal varies dramatically and certain lesions escape repair altogether and are therefore associated with high levels of mutagenicity. Central to the multistep mechanism of damage recognition in NER is the sensing of lesion-induced thermodynamic and structural alterations of DNA by the XPC-RAD23B protein and the verification of the damage by the transcription/repair factor TFIIH. Additional factors contribute to the process: UV-DDB, for the recognition of certain UV-induced lesions in particular in the context of chromatin, while the XPA protein is believed to have a role in damage verification and NER complex assembly. Here we consider the molecular mechanisms that determine repair efficiency in GG-NER based on recent structural, computational, biochemical, cellular and single molecule studies of XPC-RAD23B and its yeast ortholog Rad4. We discuss how the actions of XPC-RAD23B are integrated with those of other NER proteins and, based on recent high-resolution structures of TFIIH, present a structural model of how XPC-RAD23B and TFIIH cooperate in damage recognition and verification.
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Affiliation(s)
- Hong Mu
- Department of Biology, New York University, New York, NY 10003, USA
| | | | - Suse Broyde
- Department of Biology, New York University, New York, NY 10003, USA
| | - Jung-Eun Yeo
- Center for Genomic Integrity, Institute for Basic Science, Ulsan, Republic of Korea
| | - Orlando D Schärer
- Center for Genomic Integrity, Institute for Basic Science, Ulsan, Republic of Korea; Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea.
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30
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Rimel JK, Taatjes DJ. The essential and multifunctional TFIIH complex. Protein Sci 2018; 27:1018-1037. [PMID: 29664212 PMCID: PMC5980561 DOI: 10.1002/pro.3424] [Citation(s) in RCA: 101] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2018] [Revised: 04/04/2018] [Accepted: 04/05/2018] [Indexed: 12/19/2022]
Abstract
TFIIH is a 10‐subunit complex that regulates RNA polymerase II (pol II) transcription but also serves other important biological roles. Although much remains unknown about TFIIH function in eukaryotic cells, much progress has been made even in just the past few years, due in part to technological advances (e.g. cryoEM and single molecule methods) and the development of chemical inhibitors of TFIIH enzymes. This review focuses on the major cellular roles for TFIIH, with an emphasis on TFIIH function as a regulator of pol II transcription. We describe the structure of TFIIH and its roles in pol II initiation, promoter‐proximal pausing, elongation, and termination. We also discuss cellular roles for TFIIH beyond transcription (e.g. DNA repair, cell cycle regulation) and summarize small molecule inhibitors of TFIIH and diseases associated with defects in TFIIH structure and function.
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Affiliation(s)
- Jenna K Rimel
- Department of Chemistry & Biochemistry, University of Colorado, Boulder, Colorado, 80303
| | - Dylan J Taatjes
- Department of Chemistry & Biochemistry, University of Colorado, Boulder, Colorado, 80303
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31
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Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells. PLoS One 2018; 13:e0193677. [PMID: 29649215 PMCID: PMC5896905 DOI: 10.1371/journal.pone.0193677] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2017] [Accepted: 02/13/2018] [Indexed: 02/07/2023] Open
Abstract
Some epidemiological studies indicate that the use of mobile phones causes cancer in humans (in particular glioblastomas). It is known that DNA damage plays a key role in malignant transformation; therefore, we investigated the impact of the UMTS signal which is widely used in mobile telecommunications, on DNA stability in ten different human cell lines (six brain derived cell lines, lymphocytes, fibroblasts, liver and buccal tissue derived cells) under conditions relevant for users (SAR 0.25 to 1.00 W/kg). We found no evidence for induction of damage in single cell gel electrophoresis assays when the cells were cultivated with serum. However, clear positive effects were seen in a p53 proficient glioblastoma line (U87) when the cells were grown under serum free conditions, while no effects were found in p53 deficient glioblastoma cells (U251). Further experiments showed that the damage disappears rapidly in U87 and that exposure induced nucleotide excision repair (NER) and does not cause double strand breaks (DSBs). The observation of NER induction is supported by results of a proteome analysis indicating that several proteins involved in NER are up-regulated after exposure to UMTS; additionally, we found limited evidence for the activation of the γ-interferon pathway. The present findings show that the signal causes transient genetic instability in glioma derived cells and activates cellular defense systems.
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32
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Abstract
Although the links between defects in DNA repair and cancer are well established, an accumulating body of evidence suggests a series of functional links between genome maintenance pathways, lifespan regulation mechanisms and age-related diseases in mammals. Indeed, the growing number of DNA repair-deficient patients with progeria suggests that persistent DNA damage and genome caretakers are tightly linked to lifespan regulating circuits and age-related diseases. Here, we discuss the impact of irreparable DNA damage events in mammalian physiology highlighting the relevance of DNA repair factors in mammalian development and aging.
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33
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ASH1L histone methyltransferase regulates the handoff between damage recognition factors in global-genome nucleotide excision repair. Nat Commun 2017; 8:1333. [PMID: 29109511 PMCID: PMC5673894 DOI: 10.1038/s41467-017-01080-8] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2017] [Accepted: 08/07/2017] [Indexed: 11/09/2022] Open
Abstract
Global-genome nucleotide excision repair (GG-NER) prevents ultraviolet (UV) light-induced skin cancer by removing mutagenic cyclobutane pyrimidine dimers (CPDs). These lesions are formed abundantly on DNA wrapped around histone octamers in nucleosomes, but a specialized damage sensor known as DDB2 ensures that they are accessed by the XPC initiator of GG-NER activity. We report that DDB2 promotes CPD excision by recruiting the histone methyltransferase ASH1L, which methylates lysine 4 of histone H3. In turn, methylated H3 facilitates the docking of the XPC complex to nucleosomal histone octamers. Consequently, DDB2, ASH1L and XPC proteins co-localize transiently on histone H3-methylated nucleosomes of UV-exposed cells. In the absence of ASH1L, the chromatin binding of XPC is impaired and its ability to recruit downstream GG-NER effectors diminished. Also, ASH1L depletion suppresses CPD excision and confers UV hypersensitivity. These findings show that ASH1L configures chromatin for the effective handoff between damage recognition factors during GG-NER activity.
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34
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Rüthemann P, Balbo Pogliano C, Codilupi T, Garajovà Z, Naegeli H. Chromatin remodeler CHD1 promotes XPC-to-TFIIH handover of nucleosomal UV lesions in nucleotide excision repair. EMBO J 2017; 36:3372-3386. [PMID: 29018037 DOI: 10.15252/embj.201695742] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2016] [Revised: 08/10/2017] [Accepted: 09/08/2017] [Indexed: 12/27/2022] Open
Abstract
Ultraviolet (UV) light induces mutagenic cyclobutane pyrimidine dimers (CPDs) in nucleosomal DNA that is tightly wrapped around histone octamers. How global-genome nucleotide excision repair (GG-NER) processes CPDs despite that this chromatin arrangement is poorly understood. An increased chromatin association of CHD1 (chromodomain helicase DNA-binding 1) upon UV irradiation indicated possible roles of this chromatin remodeler in the UV damage response. Immunoprecipitation of chromatin fragments revealed that CHD1 co-localizes in part with GG-NER factors. Chromatin fractionation showed that the UV-dependent recruitment of CHD1 occurs to UV lesions in histone-assembled nucleosomal DNA and that this CHD1 relocation requires the lesion sensor XPC (xeroderma pigmentosum group C). In situ immunofluorescence analyses further demonstrate that CHD1 facilitates substrate handover from XPC to the downstream TFIIH (transcription factor IIH). Consequently, CHD1 depletion slows down CPD excision and sensitizes cells to UV-induced cytotoxicity. The finding of a CHD1-driven lesion handover between sequentially acting GG-NER factors on nucleosomal histone octamers suggests that chromatin provides a recognition scaffold enabling the detection of a subset of CPDs.
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Affiliation(s)
- Peter Rüthemann
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Chiara Balbo Pogliano
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Tamara Codilupi
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Zuzana Garajovà
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
| | - Hanspeter Naegeli
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland
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35
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Manandhar M, Lowery MG, Boulware KS, Lin KH, Lu Y, Wood RD. Transcriptional consequences of XPA disruption in human cell lines. DNA Repair (Amst) 2017; 57:76-90. [PMID: 28704716 PMCID: PMC5731452 DOI: 10.1016/j.dnarep.2017.06.028] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2017] [Revised: 06/26/2017] [Accepted: 06/27/2017] [Indexed: 11/25/2022]
Abstract
Nucleotide excision repair (NER) in mammalian cells requires the xeroderma pigmentosum group A protein (XPA) as a core factor. Remarkably, XPA and other NER proteins have been detected by chromatin immunoprecipitation at some active promoters, and NER deficiency is reported to influence the activated transcription of selected genes. However, the global influence of XPA on transcription in human cells has not been determined. We analyzed the human transcriptome by RNA sequencing (RNA-Seq). We first confirmed that XPA is confined to the cell nucleus even in the absence of external DNA damage, in contrast to previous reports that XPA is normally resident in the cytoplasm and is imported following DNA damage. We then analyzed four genetically matched human cell line pairs deficient or proficient in XPA. Of the ∼14,000 genes transcribed in each cell line, 325 genes (2%) had a significant XPA-dependent directional change in gene expression that was common to all four pairs (with a false discovery rate of 0.05). These genes were enriched in pathways for the maintenance of mitochondria. Only 27 common genes were different by more than 1.5-fold. The most significant hits were AKR1C1 and AKR1C2, involved in steroid hormone metabolism. AKR1C2 protein was lower in all of the immortalized XPA-deficient cells. Retinoic acid treatment led to modest XPA-dependent activation of some genes with transcription-related functions. We conclude that XPA status does not globally influence human gene transcription. However, XPA significantly influences expression of a small subset of genes important for mitochondrial functions and steroid hormone metabolism. The results may help explain defects in neurological function and sterility in individuals with xeroderma pigmentosum.
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Affiliation(s)
- Mandira Manandhar
- Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, P.O. Box 389, Smithville, TX, 78957, USA; MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, TX, USA
| | - Megan G Lowery
- Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, P.O. Box 389, Smithville, TX, 78957, USA
| | - Karen S Boulware
- Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, P.O. Box 389, Smithville, TX, 78957, USA
| | - Kevin H Lin
- Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, P.O. Box 389, Smithville, TX, 78957, USA
| | - Yue Lu
- Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, P.O. Box 389, Smithville, TX, 78957, USA
| | - Richard D Wood
- Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, P.O. Box 389, Smithville, TX, 78957, USA; MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, TX, USA.
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36
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Alekseev S, Nagy Z, Sandoz J, Weiss A, Egly JM, Le May N, Coin F. Transcription without XPB Establishes a Unified Helicase-Independent Mechanism of Promoter Opening in Eukaryotic Gene Expression. Mol Cell 2017; 65:504-514.e4. [DOI: 10.1016/j.molcel.2017.01.012] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2016] [Revised: 11/07/2016] [Accepted: 01/06/2017] [Indexed: 10/20/2022]
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37
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Zhu Q, Wani AA. Nucleotide Excision Repair: Finely Tuned Molecular Orchestra of Early Pre-incision Events. Photochem Photobiol 2016; 93:166-177. [PMID: 27696486 DOI: 10.1111/php.12647] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2016] [Accepted: 09/15/2016] [Indexed: 12/14/2022]
Abstract
Nucleotide excision repair (NER) eliminates a broad variety of helix-distorting DNA lesions that can otherwise cause genomic instability. NER comprises two distinct subpathways: global genomic NER (GG-NER) operating throughout the genome, and transcription-coupled NER (TC-NER) preferentially removing DNA lesions from transcribing DNA strands of transcriptionally active genes. Several NER factors undergo post-translational modifications, including ubiquitination, occurring swiftly and reversibly at DNA lesion sites. Accumulating evidence indicates that ubiquitination not only orchestrates the spatio-temporal recruitment of key protein factors to DNA lesion sites but also the productive assembly of NER pre-incision complex. This review will be restricted to the latest conceptual understanding of ubiquitin-mediated regulation of initial damage sensors of NER, that is DDB, XPC, RNAPII and CSB. We project hypothetical NER models in which ubiquitin-specific segregase, valosin-containing protein (VCP)/p97, plays an essential role in timely extraction of the congregated DNA damage sensors to functionally facilitate the DNA lesion elimination from the genome.
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Affiliation(s)
- Qianzheng Zhu
- Department of Radiology, The Ohio State University, Columbus, OH
| | - Altaf A Wani
- Department of Radiology, The Ohio State University, Columbus, OH.,Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH.,James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH
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38
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Song J, Kemp MG, Choi JH. Detection of the Excised, Damage-containing Oligonucleotide Products of Nucleotide Excision Repair in Human Cells. Photochem Photobiol 2016; 93:192-198. [PMID: 27634428 PMCID: PMC5315615 DOI: 10.1111/php.12638] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2016] [Accepted: 08/10/2016] [Indexed: 11/29/2022]
Abstract
The human nucleotide excision repair system targets a wide variety of DNA adducts for removal from DNA, including photoproducts induced by UV wavelengths of sunlight. A key feature of nucleotide excision repair is its dual incision mechanism, which results in generation of a small, damage‐containing oligonucleotide approximately 24 to 32 nt in length. Detection of these excised oligonucleotides using cell‐free extracts and purified proteins with defined DNA substrates has provided a robust biochemical assay for excision repair activity in vitro. However, the relevance of a number of in vitro findings to excision repair in living cells in vivo has remained unresolved. Over the past few years, novel methods for detecting and isolating the excised oligonucleotide products of repair in vivo have therefore been developed. Here we provide a basic outline of a sensitive and versatile in vivo excision assay and discuss how the assay both confirms previous in vitro findings and offers a number of advantages over existing cell‐based DNA repair assays. Thus, the in vivo excision assay offers a powerful tool for readily monitoring the repair of DNA lesions induced by a large number of environmental carcinogens and anticancer compounds.
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Affiliation(s)
- Jimyeong Song
- Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon, Korea.,Department of Bio-Analytical Science, University of Science & Technology, Daejeon, Korea
| | - Michael G Kemp
- Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, OH
| | - Jun-Hyuk Choi
- Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon, Korea.,Department of Bio-Analytical Science, University of Science & Technology, Daejeon, Korea
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39
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Kemp MG, Hu J. PostExcision Events in Human Nucleotide Excision Repair. Photochem Photobiol 2016; 93:178-191. [PMID: 27645806 DOI: 10.1111/php.12641] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2016] [Accepted: 08/26/2016] [Indexed: 12/27/2022]
Abstract
The nucleotide excision repair system removes a wide variety of DNA lesions from the human genome, including photoproducts induced by ultraviolet (UV) wavelengths of sunlight. A defining feature of nucleotide excision repair is its dual incision mechanism, in which two nucleolytic incision events on the damaged strand of DNA at sites bracketing the lesion generate a damage-containing DNA oligonucleotide and a single-stranded DNA gap approximately 30 nucleotides in length. Although the early events of nucleotide excision repair, which include lesion recognition and the dual incisions, have been explored in detail and are reasonably well understood, the fate of the single-stranded DNA gaps and excised oligonucleotide products of repair have not been as extensively examined. In this review, recent findings that address these less-explored aspects of nucleotide excision repair are discussed and support the concept that postincision gap and excised oligonucleotide processing are critical steps in the cellular response to DNA damage induced by UV light and other environmental carcinogens. Defects in these latter stages of repair lead to cell death and other DNA damage signaling responses and may therefore contribute to a number of human disease states associated with exposure to UV wavelengths of sunlight, including skin cancer, aging and autoimmunity.
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Affiliation(s)
- Michael G Kemp
- Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, OH
| | - Jinchuan Hu
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC
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Mallet JD, Dorr MM, Drigeard Desgarnier MC, Bastien N, Gendron SP, Rochette PJ. Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes. PLoS One 2016; 11:e0162212. [PMID: 27611318 PMCID: PMC5017652 DOI: 10.1371/journal.pone.0162212] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Accepted: 08/18/2016] [Indexed: 01/13/2023] Open
Abstract
Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced.
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Affiliation(s)
- Justin D. Mallet
- Axe Médecine Régénératrice, Centre de Recherche du CHU de Québec–Université Laval, Hôpital du Saint-Sacrement, Québec City, Québec, Canada
- Centre de Recherche en Organogénèse Expérimentale de l'Université Laval/LOEX, Université Laval, Québec City, Québec, Canada
- Département d’Ophtalmologie et ORL—chirurgie cervico-faciale, Université Laval, Québec City, Québec, Canada
| | - Marie M. Dorr
- Axe Médecine Régénératrice, Centre de Recherche du CHU de Québec–Université Laval, Hôpital du Saint-Sacrement, Québec City, Québec, Canada
- Centre de Recherche en Organogénèse Expérimentale de l'Université Laval/LOEX, Université Laval, Québec City, Québec, Canada
- Département d’Ophtalmologie et ORL—chirurgie cervico-faciale, Université Laval, Québec City, Québec, Canada
| | - Marie-Catherine Drigeard Desgarnier
- Axe Médecine Régénératrice, Centre de Recherche du CHU de Québec–Université Laval, Hôpital du Saint-Sacrement, Québec City, Québec, Canada
- Centre de Recherche en Organogénèse Expérimentale de l'Université Laval/LOEX, Université Laval, Québec City, Québec, Canada
- Département d’Ophtalmologie et ORL—chirurgie cervico-faciale, Université Laval, Québec City, Québec, Canada
| | - Nathalie Bastien
- Axe Médecine Régénératrice, Centre de Recherche du CHU de Québec–Université Laval, Hôpital du Saint-Sacrement, Québec City, Québec, Canada
- Centre de Recherche en Organogénèse Expérimentale de l'Université Laval/LOEX, Université Laval, Québec City, Québec, Canada
- Département d’Ophtalmologie et ORL—chirurgie cervico-faciale, Université Laval, Québec City, Québec, Canada
| | - Sébastien P. Gendron
- Axe Médecine Régénératrice, Centre de Recherche du CHU de Québec–Université Laval, Hôpital du Saint-Sacrement, Québec City, Québec, Canada
- Centre de Recherche en Organogénèse Expérimentale de l'Université Laval/LOEX, Université Laval, Québec City, Québec, Canada
- Département d’Ophtalmologie et ORL—chirurgie cervico-faciale, Université Laval, Québec City, Québec, Canada
| | - Patrick J. Rochette
- Axe Médecine Régénératrice, Centre de Recherche du CHU de Québec–Université Laval, Hôpital du Saint-Sacrement, Québec City, Québec, Canada
- Centre de Recherche en Organogénèse Expérimentale de l'Université Laval/LOEX, Université Laval, Québec City, Québec, Canada
- Département d’Ophtalmologie et ORL—chirurgie cervico-faciale, Université Laval, Québec City, Québec, Canada
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The functional status of DNA repair pathways determines the sensitization effect to cisplatin in non-small cell lung cancer cells. Cell Oncol (Dordr) 2016; 39:511-522. [PMID: 27473273 DOI: 10.1007/s13402-016-0291-7] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/18/2016] [Indexed: 12/29/2022] Open
Abstract
PURPOSE Cisplatin can cause a variety of DNA crosslink lesions including intra-strand and inter-strand crosslinks (ICLs), which are associated with the sensitivity of cancer cells to cisplatin. Here, we aimed to assess the contribution of the Fanconi anemia (FA), homologous recombination (HR) and nucleotide excision repair (NER) pathways to cisplatin resistance in non-small cell lung cancer (NSCLC)-derived cells. METHODS The expression of FA, HR and NER pathway-associated genes was assessed by RT-qPCR and Western blotting. siRNAs were used to knock down the expression of these genes. CCK-8 and flow cytometry assays were used to assess the viability and apoptotic rate of NSCLC-derived cells, respectively. Immunofluorescence and alkaline comet assays were used to assess the repair of ICLs. RESULTS We found that acquired cisplatin-resistant NSCLC-derived A549/DR cells exhibited markedly enhanced FA and HR repair pathway capacities compared to its parental A549 cells and another independent NSCLC-derived cell line, Calu-1, which possesses a moderate innate resistance to cisplatin. siRNA-mediated silencing of the FA-associated genes FANCL and RAD18 and the HR-associated genes BRCA1 and BRCA2 significantly potentiated the sensitivity of A549/DR cells to cisplatin compared to A549 and Calu-1 cells, suggesting that the acquired cisplatin resistance in A549/DR cells may be attributed to enhanced FA and HR pathway capacities responsible for ICL repair. Although we found that expression knockdown of the NER-associated genes XPA and ERCC1 sensitized the three NSCLC-derived cell lines to cisplatin, the sensitization effect was more significant in Calu-1 cells than in A549 and A549/DR cells, implying that the innate cisplatin resistance in Calu-1 cells may result from an increased NER activity. CONCLUSIONS Our results indicate that the functional status of DNA repair pathways determine the sensitivity of NSCLC cells to cisplatin. Direct targeting of the pathway that is involved in cisplatin resistance may be an effective strategy to surmount cisplatin resistance in NSCLC.
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Compe E, Egly JM. Nucleotide Excision Repair and Transcriptional Regulation: TFIIH and Beyond. Annu Rev Biochem 2016; 85:265-90. [DOI: 10.1146/annurev-biochem-060815-014857] [Citation(s) in RCA: 109] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
- Emmanuel Compe
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université de Strasbourg, 67404 Illkirch Cedex, Commune Urbaine Strasbourg, France; ,
| | - Jean-Marc Egly
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université de Strasbourg, 67404 Illkirch Cedex, Commune Urbaine Strasbourg, France; ,
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Tsai RYL. Balancing self-renewal against genome preservation in stem cells: How do they manage to have the cake and eat it too? Cell Mol Life Sci 2016; 73:1803-23. [PMID: 26886024 PMCID: PMC5040593 DOI: 10.1007/s00018-016-2152-y] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2015] [Revised: 01/18/2016] [Accepted: 01/28/2016] [Indexed: 01/22/2023]
Abstract
Stem cells are endowed with the awesome power of self-renewal and multi-lineage differentiation that allows them to be major contributors to tissue homeostasis. Owing to their longevity and self-renewal capacity, they are also faced with a higher risk of genomic damage compared to differentiated cells. Damage on the genome, if not prevented or repaired properly, will threaten the survival of stem cells and culminate in organ failure, premature aging, or cancer formation. It is therefore of paramount importance that stem cells remain genomically stable throughout life. Given their unique biological and functional requirement, stem cells are thought to manage genotoxic stress somewhat differently from non-stem cells. The focus of this article is to review the current knowledge on how stem cells escape the barrage of oxidative and replicative DNA damage to stay in self-renewal. A clear statement on this subject should help us better understand tissue regeneration, aging, and cancer.
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Affiliation(s)
- Robert Y L Tsai
- Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University Health Science Center, 2121 W. Holcombe Blvd, Houston, TX, 77030, USA.
- Department of Molecular and Cellular Medicine, Texas A&M University Health Science Center, College Station, TX, 77843, USA.
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Rüthemann P, Balbo Pogliano C, Naegeli H. Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers. Front Genet 2016; 7:68. [PMID: 27200078 PMCID: PMC4848295 DOI: 10.3389/fgene.2016.00068] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2016] [Accepted: 04/12/2016] [Indexed: 11/13/2022] Open
Abstract
Global-genome nucleotide excision repair (GG-NER) prevents genome instability by excising a wide range of different DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV) light or intracellular side products of metabolism. As a versatile damage sensor, xeroderma pigmentosum group C (XPC) protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4(DDB2) and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4(DDB2) or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin.
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Affiliation(s)
- Peter Rüthemann
- Institute of Pharmacology and Toxicology, Vetsuisse Faculty, University of Zurich Zurich, Switzerland
| | - Chiara Balbo Pogliano
- Institute of Pharmacology and Toxicology, Vetsuisse Faculty, University of Zurich Zurich, Switzerland
| | - Hanspeter Naegeli
- Institute of Pharmacology and Toxicology, Vetsuisse Faculty, University of Zurich Zurich, Switzerland
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Abstract
Nucleotide excision repair (NER) is a highly versatile and efficient DNA repair process, which is responsible for the removal of a large number of structurally diverse DNA lesions. Its extreme broad substrate specificity ranges from DNA damages formed upon exposure to ultraviolet radiation to numerous bulky DNA adducts induced by mutagenic environmental chemicals and cytotoxic drugs used in chemotherapy. Defective NER leads to serious diseases, such as xeroderma pigmentosum (XP). Eight XP complementation groups are known of which seven (XPA-XPG) are caused by mutations in genes involved in the NER process. The eighth gene, XPV, codes for the DNA polymerase ɳ, which replicates through DNA lesions in a process called translesion synthesis (TLS). Over the past decade, detailed structural information of these DNA repair proteins involved in eukaryotic NER and TLS have emerged. These structures allow us now to understand the molecular mechanism of the NER and TLS processes in quite some detail and we have begun to understand the broad substrate specificity of NER. In this review, we aim to highlight recent advances in the process of damage recognition and repair as well as damage tolerance by the XP proteins.
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Puumalainen MR, Rüthemann P, Min JH, Naegeli H. Xeroderma pigmentosum group C sensor: unprecedented recognition strategy and tight spatiotemporal regulation. Cell Mol Life Sci 2016; 73:547-66. [PMID: 26521083 PMCID: PMC4713717 DOI: 10.1007/s00018-015-2075-z] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Revised: 10/14/2015] [Accepted: 10/15/2015] [Indexed: 12/14/2022]
Abstract
The cellular defense system known as global-genome nucleotide excision repair (GG-NER) safeguards genome stability by eliminating a plethora of structurally unrelated DNA adducts inflicted by chemical carcinogens, ultraviolet (UV) radiation or endogenous metabolic by-products. Xeroderma pigmentosum group C (XPC) protein provides the promiscuous damage sensor that initiates this versatile NER reaction through the sequential recruitment of DNA helicases and endonucleases, which in turn recognize and excise insulting base adducts. As a DNA damage sensor, XPC protein is very unique in that it (a) displays an extremely wide substrate range, (b) localizes DNA lesions by an entirely indirect readout strategy, (c) recruits not only NER factors but also multiple repair players, (d) interacts avidly with undamaged DNA, (e) also interrogates nucleosome-wrapped DNA irrespective of chromatin compaction and (f) additionally functions beyond repair as a co-activator of RNA polymerase II-mediated transcription. Many recent reports highlighted the complexity of a post-translational circuit that uses polypeptide modifiers to regulate the spatiotemporal activity of this multiuse sensor during the UV damage response in human skin. A newly emerging concept is that stringent regulation of the diverse XPC functions is needed to prioritize DNA repair while avoiding the futile processing of undamaged genes or silent genomic sequences.
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Affiliation(s)
- Marjo-Riitta Puumalainen
- Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, 8057, Zurich, Switzerland
- Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
| | - Peter Rüthemann
- Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, 8057, Zurich, Switzerland
| | - Jun-Hyun Min
- Department of Chemistry, University of Illinois at Chicago, Chicago, IL, 60607, USA.
| | - Hanspeter Naegeli
- Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, 8057, Zurich, Switzerland.
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Impact of topical application of sulfur mustard on mice skin and distant organs DNA repair enzyme signature. Toxicol Lett 2016; 241:71-81. [DOI: 10.1016/j.toxlet.2015.11.001] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Revised: 11/02/2015] [Accepted: 11/02/2015] [Indexed: 11/23/2022]
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Han C, Wani G, Zhao R, Qian J, Sharma N, He J, Zhu Q, Wang QE, Wani AA. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair. Cell Cycle 2015; 14:1103-15. [PMID: 25483071 DOI: 10.4161/15384101.2014.973740] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3' side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4(Cdt2). Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4(Cdt2) for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.
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Affiliation(s)
- Chunhua Han
- a Department of Radiology ; The Ohio State University Wexner Medical Center ; Columbus , OH USA
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Kametani Y, Takahata C, Narita T, Tanaka K, Iwai S, Kuraoka I. FEN1 participates in repair of the 5'-phosphotyrosyl terminus of DNA single-strand breaks. Carcinogenesis 2015; 37:56-62. [PMID: 26581212 DOI: 10.1093/carcin/bgv159] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Accepted: 11/07/2015] [Indexed: 11/14/2022] Open
Abstract
Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells.
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Affiliation(s)
- Yukiko Kametani
- Graduate School of Engineering Science, Osaka University Graduate School of Engineering Science, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan and
| | - Chiaki Takahata
- Graduate School of Engineering Science, Osaka University Graduate School of Engineering Science, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan and
| | - Takashi Narita
- Laboratories for Organismal Biosystems, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Kiyoji Tanaka
- Laboratories for Organismal Biosystems, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Shigenori Iwai
- Graduate School of Engineering Science, Osaka University Graduate School of Engineering Science, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan and
| | - Isao Kuraoka
- Graduate School of Engineering Science, Osaka University Graduate School of Engineering Science, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan and
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Wang QE. DNA damage responses in cancer stem cells: Implications for cancer therapeutic strategies. World J Biol Chem 2015; 6:57-64. [PMID: 26322164 PMCID: PMC4549769 DOI: 10.4331/wjbc.v6.i3.57] [Citation(s) in RCA: 66] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/07/2015] [Revised: 04/16/2015] [Accepted: 05/28/2015] [Indexed: 02/05/2023] Open
Abstract
The identification of cancer stem cells (CSCs) that are responsible for tumor initiation, growth, metastasis, and therapeutic resistance might lead to a new thinking on cancer treatments. Similar to stem cells, CSCs also display high resistance to radiotherapy and chemotherapy with genotoxic agents. Thus, conventional therapy may shrink the tumor volume but cannot eliminate cancer. Eradiation of CSCs represents a novel therapeutic strategy. CSCs possess a highly efficient DNA damage response (DDR) system, which is considered as a contributor to the resistance of these cells from exposures to DNA damaging agents. Targeting of enhanced DDR in CSCs is thus proposed to facilitate the eradication of CSCs by conventional therapeutics. To achieve this aim, a better understanding of the cellular responses to DNA damage in CSCs is needed. In addition to the protein kinases and enzymes that are involved in DDR, other processes that affect the DDR including chromatin remodeling should also be explored.
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