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1
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Ceccaldi R, Cejka P. Mechanisms and regulation of DNA end resection in the maintenance of genome stability. Nat Rev Mol Cell Biol 2025:10.1038/s41580-025-00841-4. [PMID: 40133633 DOI: 10.1038/s41580-025-00841-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/24/2025] [Indexed: 03/27/2025]
Abstract
DNA end resection is a crucial early step in most DNA double-strand break (DSB) repair pathways. Resection involves the nucleolytic degradation of 5' ends at DSB sites to generate 3' single-stranded DNA overhangs. The first, short-range resection step is catalysed by the nuclease MRE11, acting as part of the MRE11-RAD50-NBS1 complex. Subsequent long-range resection is catalysed by the nucleases EXO1 and/or DNA2. Resected DNA is necessary for homology search and the priming of DNA synthesis in homologous recombination. DNA overhangs may also mediate DNA annealing in the microhomology-mediated end-joining and single-strand annealing pathways, and activate the DNA damage response. By contrast, DNA end resection inhibits DSB repair by non-homologous end-joining. In this Review, we discuss the importance of DNA end resection in various DSB repair pathways, the molecular mechanisms of end resection and its regulation, focusing on phosphorylation and other post-translational modifications that control resection throughout the cell cycle and in response to DNA damage.
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Affiliation(s)
- Raphael Ceccaldi
- INSERM U830, PSL Research University, Institut Curie, Paris, France.
| | - Petr Cejka
- Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland.
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2
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Paliwal S, Dey P, Tambat S, Shinohara A, Mehta G. Role of ATP-dependent chromatin remodelers in meiosis. Trends Genet 2025; 41:236-250. [PMID: 39550320 DOI: 10.1016/j.tig.2024.10.004] [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: 06/26/2024] [Revised: 09/29/2024] [Accepted: 10/21/2024] [Indexed: 11/18/2024]
Abstract
In eukaryotic cells, DNA is wrapped around histone octamers to compact the genome. Although such compaction is required for the precise segregation of the genome during cell division, it restricts the DNA-protein interactions essential for several cellular processes. During meiosis, a specialized cell division process that produces gametes, several DNA-protein interactions are crucial for assembling meiosis-specific chromosome structures, meiotic recombination, chromosome segregation, and transcriptional regulation. The role of chromatin remodelers (CRs) in facilitating DNA-protein transactions during mitosis is well appreciated, whereas how they facilitate meiosis-specific processes is poorly understood. In this review, we summarize experimental evidence supporting the role of CRs in meiosis in various model systems and suggest future perspectives to advance the field.
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Affiliation(s)
- Sheetal Paliwal
- Laboratory of Chromosome Dynamics and Gene Regulation, Department of Biotechnology, Indian Institute of Technology, Hyderabad, India
| | - Partha Dey
- Laboratory of Chromosome Dynamics and Gene Regulation, Department of Biotechnology, Indian Institute of Technology, Hyderabad, India
| | - Swarangi Tambat
- Laboratory of Chromosome Dynamics and Gene Regulation, Department of Biotechnology, Indian Institute of Technology, Hyderabad, India
| | - Akira Shinohara
- Institute for Protein Research, University of Osaka, Osaka, Japan
| | - Gunjan Mehta
- Laboratory of Chromosome Dynamics and Gene Regulation, Department of Biotechnology, Indian Institute of Technology, Hyderabad, India.
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3
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Hung SH, Liang Y, Heyer WD. Multifaceted roles of H2B mono-ubiquitylation in D-loop metabolism during homologous recombination repair. Nucleic Acids Res 2025; 53:gkaf081. [PMID: 39970303 PMCID: PMC11822380 DOI: 10.1093/nar/gkaf081] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2024] [Revised: 01/21/2025] [Accepted: 01/29/2025] [Indexed: 02/16/2025] Open
Abstract
Repairing DNA double-strand breaks is crucial for maintaining genome integrity, which occurs primarily through homologous recombination (HR) in Saccharomyces cerevisiae. Nucleosomes, composed of DNA wrapped around a histone octamer, present a natural barrier to end resection to initiate HR, but the impact on the downstream HR steps of homology search, DNA strand invasion, and repair synthesis remain to be determined. Displacement loops (D-loops) play a pivotal role in HR, yet the influence of chromatin dynamics on D-loop metabolism remains unclear. Using the physical D-loop capture and D-loop extension (DLE) assays to track HR intermediates, we employed genetic analysis to reveal that H2B mono-ubiquitylation (H2Bubi) affects multiple steps during HR repair. We infer that H2Bubi modulates chromatin structure, not only promoting histone degradation for nascent D-loop formation but also stabilizing extended D-loops through nucleosome assembly. Furthermore, H2Bubi regulates DNA resection via Rad9 recruitment to suppress a feedback control mechanism that dampens D-loop formation and DLE at hyper-resected ends. Through physical and genetic assays to determine repair outcomes, we demonstrate that H2Bubi plays a crucial role in preventing break-induced replication and thus promoting genomic stability.
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Affiliation(s)
- Shih-Hsun Hung
- Department of Microbiology and Molecular Genetics, University of California, Davis, One Shields Ave, Davis, CA 95616, United States
| | - Yuan Liang
- Department of Microbiology and Molecular Genetics, University of California, Davis, One Shields Ave, Davis, CA 95616, United States
| | - Wolf-Dietrich Heyer
- Department of Microbiology and Molecular Genetics, University of California, Davis, One Shields Ave, Davis, CA 95616, United States
- Department of Molecular and Cellular Biology, University of California, Davis, One Shields Ave, Davis, CA 95616, United States
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4
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Huang PC, Hong S, Alnaser HF, Mimitou EP, Kim KP, Murakami H, Keeney S. Meiotic DNA break resection and recombination rely on chromatin remodeler Fun30. EMBO J 2025; 44:200-224. [PMID: 39613969 PMCID: PMC11695836 DOI: 10.1038/s44318-024-00318-8] [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: 04/30/2024] [Revised: 10/03/2024] [Accepted: 11/12/2024] [Indexed: 12/01/2024] Open
Abstract
DNA double-strand breaks (DSBs) are nucleolytically processed to generate single-stranded DNA for homologous recombination. In Saccharomyces cerevisiae meiosis, this resection involves nicking by the Mre11-Rad50-Xrs2 complex (MRX), then exonucleolytic digestion by Exo1. Chromatin remodeling at meiotic DSBs is thought necessary for resection, but the remodeling enzyme was unknown. Here we show that the SWI/SNF-like ATPase Fun30 plays a major, nonredundant role in meiotic resection. A fun30 mutation shortened resection tracts almost as severely as an exo1-nd (nuclease-dead) mutation, and resection was further shortened in a fun30 exo1-nd double mutant. Fun30 associates with chromatin in response to DSBs, and the constitutive positioning of nucleosomes governs resection endpoint locations in the absence of Fun30. We infer that Fun30 promotes both the MRX- and Exo1-dependent steps in resection, possibly by removing nucleosomes from broken chromatids. Moreover, the extremely short resection in fun30 exo1-nd double mutants is accompanied by compromised interhomolog recombination bias, leading to defects in recombination and chromosome segregation. Thus, this study also provides insight about the minimal resection lengths needed for robust recombination.
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Affiliation(s)
- Pei-Ching Huang
- Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY, 10021, USA
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
- Metagenomi, Emeryville, CA, 94608, USA
| | - Soogil Hong
- Department of Life Science, Chung-Ang University, Seoul, 06974, South Korea
| | - Hasan F Alnaser
- Chromosome and Cellular Dynamics Section, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, UK
| | - Eleni P Mimitou
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
- Immunai, 430 E 29th St, New York, NY, 10016, USA
| | - Keun P Kim
- Department of Life Science, Chung-Ang University, Seoul, 06974, South Korea
- Research Center for Biomolecules and Biosystems, Chung-Ang University, Seoul, 06974, South Korea
| | - Hajime Murakami
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.
- Chromosome and Cellular Dynamics Section, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, UK.
| | - Scott Keeney
- Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY, 10021, USA.
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.
- Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.
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5
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Constantinou M, Charidemou E, Shanlitourk I, Strati K, Kirmizis A. Yeast Nat4 regulates DNA damage checkpoint signaling through its N-terminal acetyltransferase activity on histone H4. PLoS Genet 2024; 20:e1011433. [PMID: 39356727 PMCID: PMC11472955 DOI: 10.1371/journal.pgen.1011433] [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: 01/30/2024] [Revised: 10/14/2024] [Accepted: 09/20/2024] [Indexed: 10/04/2024] Open
Abstract
The DNA damage response (DDR) constitutes a vital cellular process that safeguards genome integrity. This biological process involves substantial alterations in chromatin structure, commonly orchestrated by epigenetic enzymes. Here, we show that the epigenetic modifier N-terminal acetyltransferase 4 (Nat4), known to acetylate the alpha-amino group of serine 1 on histones H4 and H2A, is implicated in the response to DNA damage in S. cerevisiae. Initially, we demonstrate that yeast cells lacking Nat4 have an increased sensitivity to DNA damage and accumulate more DNA breaks than wild-type cells. Accordingly, upon DNA damage, NAT4 gene expression is elevated, and the enzyme is specifically recruited at double-strand breaks. Delving deeper into its effects on the DNA damage signaling cascade, nat4-deleted cells exhibit lower levels of the damage-induced modification H2AS129ph (γH2A), accompanied by diminished binding of the checkpoint control protein Rad9 surrounding the double-strand break. Consistently, Mec1 kinase recruitment at double-strand breaks, critical for H2AS129ph deposition and Rad9 retention, is significantly impaired in nat4Δ cells. Consequently, Mec1-dependent phosphorylation of downstream effector kinase Rad53, indicative of DNA damage checkpoint activation, is reduced. Importantly, we found that the effects of Nat4 in regulating the checkpoint signaling cascade are mediated by its N-terminal acetyltransferase activity targeted specifically towards histone H4. Overall, this study points towards a novel functional link between histone N-terminal acetyltransferase Nat4 and the DDR, associating a new histone-modifying activity in the maintenance of genome integrity.
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Affiliation(s)
| | - Evelina Charidemou
- Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus
| | - Izge Shanlitourk
- Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus
| | - Katerina Strati
- Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus
| | - Antonis Kirmizis
- Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus
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Hung SH, Liang Y, Heyer WD. Multifaceted roles of H2B mono-ubiquitylation in D-loop metabolism during homologous recombination repair. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.09.13.612919. [PMID: 39314463 PMCID: PMC11419151 DOI: 10.1101/2024.09.13.612919] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/25/2024]
Abstract
Repairing DNA double-strand breaks is crucial for maintaining genome integrity, which occurs primarily through homologous recombination (HR) in S. cerevisiae. Nucleosomes, composed of DNA wrapped around a histone octamer, present a natural barrier to end-resection to initiate HR, but the impact on the downstream HR steps of homology search, DNA strand invasion and repair synthesis remain to be determined. Displacement loops (D-loops) play a pivotal role in HR, yet the influence of chromatin dynamics on D-loop metabolism remains unclear. Using the physical D-loop capture (DLC) and D-loop extension (DLE) assays to track HR intermediates, we employed genetic analysis to reveal that H2B mono-ubiquitylation (H2Bubi) affects multiple steps during HR repair. We infer that H2Bubi modulates chromatin structure, not only promoting histone degradation for nascent D-loop formation but also stabilizing extended D-loops through nucleosome assembly. Furthermore, H2Bubi regulates DNA resection via Rad9 recruitment to suppress a feedback control mechanism that dampens D-loop formation and extension at hyper-resected ends. Through physical and genetic assays to determine repair outcomes, we demonstrate that H2Bubi plays a crucial role in preventing break-induced replication and thus promoting genomic stability. Highlights H2Bubi is epistatic to H2A.Z and INO80 in promoting homology search and D-loop formationH2Bubi stabilizes extended D-loopExcessive resection counteracts D-loop formation and extensionH2Bubi promotes crossover events and limits the frequency of break-induced replication outcomes in HR repair.
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7
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Wang X, Zhao X, Yu Z, Fan T, Guo Y, Liang J, Wang Y, Zhan J, Chen G, Zhou C, Zhang X, Li X, Chen X. Rtt105 stimulates Rad51-ssDNA assembly and orchestrates Rad51 and RPA actions to promote homologous recombination repair. Proc Natl Acad Sci U S A 2024; 121:e2402262121. [PMID: 39145931 PMCID: PMC11348298 DOI: 10.1073/pnas.2402262121] [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: 02/02/2024] [Accepted: 07/09/2024] [Indexed: 08/16/2024] Open
Abstract
Homologous recombination (HR) is essential for the maintenance of genome stability. During HR, Replication Protein A (RPA) rapidly coats the 3'-tailed single-strand DNA (ssDNA) generated by end resection. Then, the ssDNA-bound RPA must be timely replaced by Rad51 recombinase to form Rad51 nucleoprotein filaments that drive homology search and HR repair. How cells regulate Rad51 assembly dynamics and coordinate RPA and Rad51 actions to ensure proper HR remains poorly understood. Here, we identified that Rtt105, a Ty1 transposon regulator, acts to stimulate Rad51 assembly and orchestrate RPA and Rad51 actions during HR. We found that Rtt105 interacts with Rad51 in vitro and in vivo and restrains the adenosine 5' triphosphate (ATP) hydrolysis activity of Rad51. We showed that Rtt105 directly stimulates dynamic Rad51-ssDNA assembly, strand exchange, and D-loop formation in vitro. Notably, we found that Rtt105 physically regulates the binding of Rad51 and RPA to ssDNA via different motifs and that both regulations are necessary and epistatic in promoting Rad51 nucleation, strand exchange, and HR repair. Consequently, disrupting either of the interactions impaired HR and conferred DNA damage sensitivity, underscoring the importance of Rtt105 in orchestrating the actions of Rad51 and RPA. Our work reveals additional layers of mechanisms regulating Rad51 filament dynamics and the coordination of HR.
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Affiliation(s)
- Xuejie Wang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Xiaocong Zhao
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Zhengshi Yu
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Tianai Fan
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Yunjing Guo
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Jianqiang Liang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Yanyan Wang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Jingfei Zhan
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Guifang Chen
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Chun Zhou
- School of Public Health, Zhejiang University School of Medicine, Hangzhou310058, China
| | - Xinghua Zhang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Xiangpan Li
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
| | - Xuefeng Chen
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Department of Radiation Oncology, Renmin Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan430072, China
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8
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Chen R, Zhao MJ, Li YM, Liu AH, Wang RX, Mei YC, Chen X, Du HN. Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair. SCIENCE CHINA. LIFE SCIENCES 2024; 67:1089-1105. [PMID: 38842635 DOI: 10.1007/s11427-024-2543-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/25/2024] [Accepted: 01/29/2024] [Indexed: 06/07/2024]
Abstract
Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.
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Affiliation(s)
- Runfa Chen
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Center for Immunology and Metabolism, Hubei Clinical Research Center of Emergency and Resuscitation, Emergency Center of Zhongnan Hospital, Wuhan University, Wuhan, 430072, China
| | - Meng-Jie Zhao
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Center for Immunology and Metabolism, Hubei Clinical Research Center of Emergency and Resuscitation, Emergency Center of Zhongnan Hospital, Wuhan University, Wuhan, 430072, China
| | - Yu-Min Li
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Center for Immunology and Metabolism, Hubei Clinical Research Center of Emergency and Resuscitation, Emergency Center of Zhongnan Hospital, Wuhan University, Wuhan, 430072, China
| | - Ao-Hui Liu
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Center for Immunology and Metabolism, Hubei Clinical Research Center of Emergency and Resuscitation, Emergency Center of Zhongnan Hospital, Wuhan University, Wuhan, 430072, China
| | - Ru-Xin Wang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Center for Immunology and Metabolism, Hubei Clinical Research Center of Emergency and Resuscitation, Emergency Center of Zhongnan Hospital, Wuhan University, Wuhan, 430072, China
| | - Yu-Chao Mei
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Center for Immunology and Metabolism, Hubei Clinical Research Center of Emergency and Resuscitation, Emergency Center of Zhongnan Hospital, Wuhan University, Wuhan, 430072, China
| | - Xuefeng Chen
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, 430072, China
| | - Hai-Ning Du
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Center for Immunology and Metabolism, Hubei Clinical Research Center of Emergency and Resuscitation, Emergency Center of Zhongnan Hospital, Wuhan University, Wuhan, 430072, China.
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Huang PC, Hong S, Mimitou EP, Kim KP, Murakami H, Keeney S. Meiotic DNA break resection and recombination rely on chromatin remodeler Fun30. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.04.17.589955. [PMID: 38659928 PMCID: PMC11042300 DOI: 10.1101/2024.04.17.589955] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/26/2024]
Abstract
DNA double-strand breaks (DSBs) are nucleolytically processed to generate single-stranded DNA tails for homologous recombination. In Saccharomyces cerevisiae meiosis, this 5'-to-3' resection involves initial nicking by the Mre11-Rad50-Xrs2 complex (MRX) plus Sae2, then exonucleolytic digestion by Exo1. Chromatin remodeling adjacent to meiotic DSBs is thought to be necessary for resection, but the relevant remodeling activity was unknown. Here we show that the SWI/SNF-like ATPase Fun30 plays a major, non-redundant role in resecting meiotic DSBs. A fun30 null mutation shortened resection tract lengths almost as severely as an exo1-nd (nuclease-dead) mutation, and resection was further shortened in the fun30 exo1-nd double mutant. Fun30 associates with chromatin in response to meiotic DSBs, and the constitutive positioning of nucleosomes governs resection endpoint locations in the absence of Fun30. We infer that Fun30 directly promotes both the MRX- and Exo1-dependent steps in resection, possibly by removing nucleosomes from broken chromatids. Moreover, we found that the extremely short resection in the fun30 exo1-nd double mutant is accompanied by compromised interhomolog recombination bias, leading to defects in recombination and chromosome segregation. Thus, this study also provides insight about the minimal resection lengths needed for robust recombination.
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Affiliation(s)
- Pei-Ching Huang
- Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY 10021
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065
| | - Soogil Hong
- Department of Life Science, Chung-Ang University, Seoul 06974, South Korea
| | - Eleni P. Mimitou
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065
| | - Keun P. Kim
- Department of Life Science, Chung-Ang University, Seoul 06974, South Korea
- Research Center for Biomolecules and Biosystems, Chung-Ang University, Seoul 06974, South Korea
| | - Hajime Murakami
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065
| | - Scott Keeney
- Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY 10021
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065
- Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065
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10
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Sachs P, Bergmaier P, Treutwein K, Mermoud JE. The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse. Genes (Basel) 2023; 14:1793. [PMID: 37761933 PMCID: PMC10530723 DOI: 10.3390/genes14091793] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Revised: 09/08/2023] [Accepted: 09/08/2023] [Indexed: 09/29/2023] Open
Abstract
In vertebrates, SMARCAD1 participates in transcriptional regulation, heterochromatin maintenance, DNA repair, and replication. The molecular basis underlying its involvement in these processes is not well understood. We identified the RNA polymerase III general transcription factor TFIIIC as an interaction partner of native SMARCAD1 in mouse and human models using endogenous co-immunoprecipitations. TFIIIC has dual functionality, acting as a general transcription factor and as a genome organizer separating chromatin domains. We found that its partnership with SMARCAD1 is conserved across different mammalian cell types, from somatic to pluripotent cells. Using purified proteins, we confirmed that their interaction is direct. A gene expression analysis suggested that SMARCAD1 is dispensable for TFIIIC function as an RNA polymerase III transcription factor in mouse ESCs. The distribution of TFIIIC and SMARCAD1 in the ESC genome is distinct, and unlike in yeast, SMARCAD1 is not enriched at active tRNA genes. Further analysis of SMARCAD1-binding partners in pluripotent and differentiated mammalian cells reveals that SMARCAD1 associates with several factors that have key regulatory roles in chromatin organization, such as cohesin, laminB, and DDX5. Together, our work suggests for the first time that the SMARCAD1 enzyme participates in genome organization in mammalian nuclei through interactions with architectural proteins.
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Affiliation(s)
- Parysatis Sachs
- Institute of Molecular Biology and Tumor Research, Philipps University Marburg, 35043 Marburg, Germany
- CMC Development, R&D, Sanofi, 65926 Frankfurt, Germany
| | - Philipp Bergmaier
- Institute of Molecular Biology and Tumor Research, Philipps University Marburg, 35043 Marburg, Germany
- Global Development Operations, R&D, Merck Healthcare, 64293 Darmstadt, Germany
| | - Katrin Treutwein
- Institute of Molecular Biology and Tumor Research, Philipps University Marburg, 35043 Marburg, Germany
| | - Jacqueline E. Mermoud
- Institute of Molecular Biology and Tumor Research, Philipps University Marburg, 35043 Marburg, Germany
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11
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Karl LA, Galanti L, Bantele SC, Metzner F, Šafarić B, Rajappa L, Foster B, Pires VB, Bansal P, Chacin E, Basquin J, Duderstadt KE, Kurat CF, Bartke T, Hopfner KP, Pfander B. A SAM-key domain required for enzymatic activity of the Fun30 nucleosome remodeler. Life Sci Alliance 2023; 6:e202201790. [PMID: 37468166 PMCID: PMC10355287 DOI: 10.26508/lsa.202201790] [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: 10/31/2022] [Revised: 06/28/2023] [Accepted: 06/29/2023] [Indexed: 07/21/2023] Open
Abstract
Fun30 is the prototype of the Fun30-SMARCAD1-ETL subfamily of nucleosome remodelers involved in DNA repair and gene silencing. These proteins appear to act as single-subunit nucleosome remodelers, but their molecular mechanisms are, at this point, poorly understood. Using multiple sequence alignment and structure prediction, we identify an evolutionarily conserved domain that is modeled to contain a SAM-like fold with one long, protruding helix, which we term SAM-key. Deletion of the SAM-key within budding yeast Fun30 leads to a defect in DNA repair and gene silencing similar to that of the fun30Δ mutant. In vitro, Fun30 protein lacking the SAM-key is able to bind nucleosomes but is deficient in DNA-stimulated ATPase activity and nucleosome sliding and eviction. A structural model based on AlphaFold2 prediction and verified by crosslinking-MS indicates an interaction of the long SAM-key helix with protrusion I, a subdomain located between the two ATPase lobes that is critical for control of enzymatic activity. Mutation of the interaction interface phenocopies the domain deletion with a lack of DNA-stimulated ATPase activation and a nucleosome-remodeling defect, thereby confirming a role of the SAM-key helix in regulating ATPase activity. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, thus highlighting the importance of allosteric activation for this class of enzymes.
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Affiliation(s)
- Leonhard A Karl
- DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Lorenzo Galanti
- DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
- Genome Maintenance Mechanisms in Health and Disease, Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany
- Genome Maintenance Mechanisms in Health and Disease, Institute of Genome Stability in Ageing and Disease, CECAD Research Center, University of Cologne, Cologne, Germany
| | - Susanne Cs Bantele
- DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Felix Metzner
- Gene Center, Department of Biochemistry, Ludwig-Maximilians-Universität, Munich, Germany
| | - Barbara Šafarić
- Structure and Dynamics of Molecular Machines, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Lional Rajappa
- Structure and Dynamics of Molecular Machines, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Benjamin Foster
- Institute of Functional Epigenetics (IFE), Helmholtz Zentrum München, Neuherberg, Germany
| | - Vanessa Borges Pires
- Genome Maintenance Mechanisms in Health and Disease, Institute of Genome Stability in Ageing and Disease, CECAD Research Center, University of Cologne, Cologne, Germany
| | - Priyanka Bansal
- Biomedical Center Munich (BMC), Division of Molecular Biology, Faculty of Medicine, Ludwig-Maximilians-Universität in Munich, Martinsried, Germany
| | - Erika Chacin
- Biomedical Center Munich (BMC), Division of Molecular Biology, Faculty of Medicine, Ludwig-Maximilians-Universität in Munich, Martinsried, Germany
| | - Jerôme Basquin
- Crystallization Facility, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Karl E Duderstadt
- Structure and Dynamics of Molecular Machines, Max Planck Institute of Biochemistry, Martinsried, Germany
- Physik Department, Technische Universität München, Munich, Germany
| | - Christoph F Kurat
- Biomedical Center Munich (BMC), Division of Molecular Biology, Faculty of Medicine, Ludwig-Maximilians-Universität in Munich, Martinsried, Germany
| | - Till Bartke
- Institute of Functional Epigenetics (IFE), Helmholtz Zentrum München, Neuherberg, Germany
| | - Karl-Peter Hopfner
- Gene Center, Department of Biochemistry, Ludwig-Maximilians-Universität, Munich, Germany
| | - Boris Pfander
- DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
- Genome Maintenance Mechanisms in Health and Disease, Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany
- Genome Maintenance Mechanisms in Health and Disease, Institute of Genome Stability in Ageing and Disease, CECAD Research Center, University of Cologne, Cologne, Germany
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12
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Oh JM, Kang Y, Park J, Sung Y, Kim D, Seo Y, Lee E, Ra J, Amarsanaa E, Park YU, Lee S, Hwang J, Kim H, Schärer O, Cho S, Lee C, Takata KI, Lee J, Myung K. MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1. Nucleic Acids Res 2023; 51:5584-5602. [PMID: 37140056 PMCID: PMC10287916 DOI: 10.1093/nar/gkad308] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2022] [Revised: 04/04/2023] [Accepted: 04/27/2023] [Indexed: 05/05/2023] Open
Abstract
DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.
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Affiliation(s)
- Jung-Min Oh
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Oral Biochemistry, Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea
| | - Yujin Kang
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Jumi Park
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Yubin Sung
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Dayoung Kim
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Yuri Seo
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Eun A Lee
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Jae Sun Ra
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Enkhzul Amarsanaa
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Young-Un Park
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Seon Young Lee
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Jung Me Hwang
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Hongtae Kim
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Orlando Schärer
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Seung Woo Cho
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Changwook Lee
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Kei-ichi Takata
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Ja Yil Lee
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Kyungjae Myung
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
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13
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Gan X, Zhang Y, Jiang D, Shi J, Zhao H, Xie C, Wang Y, Xu J, Zhang X, Cai G, Wang H, Huang J, Chen X. Proper RPA acetylation promotes accurate DNA replication and repair. Nucleic Acids Res 2023; 51:5565-5583. [PMID: 37140030 PMCID: PMC10287905 DOI: 10.1093/nar/gkad291] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2023] [Revised: 04/06/2023] [Accepted: 04/12/2023] [Indexed: 05/05/2023] Open
Abstract
The single-stranded DNA (ssDNA) binding protein complex RPA plays a critical role in promoting DNA replication and multiple DNA repair pathways. However, how RPA is regulated to achieve its functions precisely in these processes remains elusive. Here, we found that proper acetylation and deacetylation of RPA are required to regulate RPA function in promoting high-fidelity DNA replication and repair. We show that yeast RPA is acetylated on multiple conserved lysines by the acetyltransferase NuA4 upon DNA damage. Mimicking constitutive RPA acetylation or blocking its acetylation causes spontaneous mutations with the signature of micro-homology-mediated large deletions or insertions. In parallel, improper RPA acetylation/deacetylation impairs DNA double-strand break (DSB) repair by the accurate gene conversion or break-induced replication while increasing the error-prone repair by single-strand annealing or alternative end joining. Mechanistically, we show that proper acetylation and deacetylation of RPA ensure its normal nuclear localization and ssDNA binding ability. Importantly, mutation of the equivalent residues in human RPA1 also impairs RPA binding on ssDNA, leading to attenuated RAD51 loading and homologous recombination repair. Thus, timely RPA acetylation and deacetylation likely represent a conserved mechanism promoting high-fidelity replication and repair while discriminating the error-prone repair mechanisms in eukaryotes.
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Affiliation(s)
- Xiaoli Gan
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Wuhan University, Wuhan, Hubei 430072, China
| | - Yueyue Zhang
- The First Affiliated Hospital of USTC, MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, China
| | - Donghao Jiang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Wuhan University, Wuhan, Hubei 430072, China
| | - Jingyao Shi
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Wuhan University, Wuhan, Hubei 430072, China
| | - Han Zhao
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Wuhan University, Wuhan, Hubei 430072, China
| | - Chengyu Xie
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Wuhan University, Wuhan, Hubei 430072, China
| | - Yanyan Wang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Wuhan University, Wuhan, Hubei 430072, China
| | - Jingyan Xu
- Department of Hematology, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, China
| | - Xinghua Zhang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Wuhan University, Wuhan, Hubei 430072, China
| | - Gang Cai
- The First Affiliated Hospital of USTC, MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, China
| | - Hailong Wang
- Beijing Key Laboratory of DNA Damage Response and College of Life Sciences, Capital Normal University, Beijing 100048, China
| | - Jun Huang
- The MOE Key Laboratory of Biosystems Homeostasis & Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Xuefeng Chen
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Wuhan University, Wuhan, Hubei 430072, China
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14
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Liu G, Li J, He B, Yan J, Zhao J, Wang X, Zhao X, Xu J, Wu Y, Zhang S, Gan X, Zhou C, Li X, Zhang X, Chen X. Bre1/RNF20 promotes Rad51-mediated strand exchange and antagonizes the Srs2/FBH1 helicases. Nat Commun 2023; 14:3024. [PMID: 37230987 DOI: 10.1038/s41467-023-38617-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Accepted: 05/10/2023] [Indexed: 05/27/2023] Open
Abstract
Central to homologous recombination (HR) is the assembly of Rad51 recombinase on single-strand DNA (ssDNA), forming the Rad51-ssDNA filament. How the Rad51 filament is efficiently established and sustained remains partially understood. Here, we find that the yeast ubiquitin ligase Bre1 and its human homolog RNF20, a tumor suppressor, function as recombination mediators, promoting Rad51 filament formation and subsequent reactions via multiple mechanisms independent of their ligase activities. We show that Bre1/RNF20 interacts with Rad51, directs Rad51 to ssDNA, and facilitates Rad51-ssDNA filament assembly and strand exchange in vitro. In parallel, Bre1/RNF20 interacts with the Srs2 or FBH1 helicase to counteract their disrupting effect on the Rad51 filament. We demonstrate that the above functions of Bre1/RNF20 contribute to HR repair in cells in a manner additive to the mediator protein Rad52 in yeast or BRCA2 in human. Thus, Bre1/RNF20 provides an additional layer of mechanism to directly control Rad51 filament dynamics.
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Affiliation(s)
- Guangxue Liu
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Department of Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China
| | - Jimin Li
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Department of Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China
| | - Boxue He
- Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX, USA
| | - Jiaqi Yan
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Department of Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China
| | - Jingyu Zhao
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Department of Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China
| | - Xuejie Wang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Department of Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China
| | - Xiaocong Zhao
- The Institute for Advanced Studies, Wuhan University, Wuhan, China
| | - Jingyan Xu
- Department of Hematology, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, China
| | - Yeyao Wu
- School of Public Health, Zhejiang University School of Medicine, Hangzhou, China
| | - Simin Zhang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Department of Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China
| | - Xiaoli Gan
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Department of Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China
| | - Chun Zhou
- School of Public Health, Zhejiang University School of Medicine, Hangzhou, China
| | - Xiangpan Li
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Department of Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China
| | - Xinghua Zhang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Department of Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China
- The Institute for Advanced Studies, Wuhan University, Wuhan, China
| | - Xuefeng Chen
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, Department of Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China.
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15
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Polleys EJ, Del Priore I, Haber JE, Freudenreich CH. Structure-forming CAG/CTG repeats interfere with gap repair to cause repeat expansions and chromosome breaks. Nat Commun 2023; 14:2469. [PMID: 37120647 PMCID: PMC10148874 DOI: 10.1038/s41467-023-37901-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Accepted: 04/04/2023] [Indexed: 05/01/2023] Open
Abstract
Expanded CAG/CTG repeats are sites of DNA damage, leading to repeat length changes. Homologous recombination (HR) is one cause of repeat instability and we hypothesized that gap filling was a driver of repeat instability during HR. To test this, we developed an assay such that resection and ssDNA gap fill-in would occur across a (CAG)70 or (CTG)70 repeat tract. When the ssDNA template was a CTG sequence, there were increased repeat contractions and a fragile site was created leading to large-scale deletions. When the CTG sequence was on the resected strand, resection was inhibited, resulting in repeat expansions. Increased nucleolytic processing by deletion of Rad9, the ortholog of 53BP1, rescued repeat instability and chromosome breakage. Loss of Rad51 increased contractions implicating a protective role for Rad51 on ssDNA. Together, our work implicates structure-forming repeats as an impediment to resection and gap-filling which can lead to mutations and large-scale deletions.
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Affiliation(s)
- Erica J Polleys
- Department of Biology, Tufts University, Medford, MA, 02155, USA.
| | | | - James E Haber
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, 02454, USA
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16
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Gnügge R, Reginato G, Cejka P, Symington LS. Sequence and chromatin features guide DNA double-strand break resection initiation. Mol Cell 2023; 83:1237-1250.e15. [PMID: 36917982 PMCID: PMC10131398 DOI: 10.1016/j.molcel.2023.02.010] [Citation(s) in RCA: 20] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2022] [Revised: 01/09/2023] [Accepted: 02/09/2023] [Indexed: 03/14/2023]
Abstract
DNA double-strand breaks (DSBs) are cytotoxic genome lesions that must be accurately and efficiently repaired to ensure genome integrity. In yeast, the Mre11-Rad50-Xrs2 (MRX) complex nicks 5'-terminated DSB ends to initiate nucleolytic processing of DSBs for repair by homologous recombination. How MRX-DNA interactions support 5' strand-specific nicking and how nicking is influenced by the chromatin context have remained elusive. Using a deep sequencing-based assay, we mapped MRX nicks at single-nucleotide resolution next to multiple DSBs in the yeast genome. We observed that the DNA end-binding Ku70-Ku80 complex directed DSB-proximal nicks and that repetitive MRX cleavage extended the length of resection tracts. We identified a sequence motif and a DNA meltability profile that is preferentially nicked by MRX. Furthermore, we found that nucleosomes as well as transcription impeded MRX incisions. Our findings suggest that local DNA sequence and chromatin features shape the activity of this central DSB repair complex.
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Affiliation(s)
- Robert Gnügge
- Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA.
| | - Giordano Reginato
- Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), 8093 Zürich, Switzerland; Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Faculty of Biomedical Sciences, 6500 Bellinzona, Switzerland
| | - Petr Cejka
- Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), 8093 Zürich, Switzerland; Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Faculty of Biomedical Sciences, 6500 Bellinzona, Switzerland
| | - Lorraine S Symington
- Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA; Department of Genetics & Development, Columbia University Irving Medical Center, New York, NY 10032, USA.
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17
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Gao N, Dai B, Nie X, Zhao Q, Zhu W, Chen J. Fun30 nucleosome remodeller regulates white-to-opaque switching in Candida albicans. Acta Biochim Biophys Sin (Shanghai) 2023; 55:508-517. [PMID: 36896644 PMCID: PMC10160231 DOI: 10.3724/abbs.2023031] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/06/2023] Open
Abstract
Candida albicans ( C. albicans) is an opportunistic pathogen in humans and possesses a white-opaque heritable switching system. Wor1 is a master regulator of white-opaque switching and is essential for opaque cell formation in C. albicans. However, the regulatory network of Wor1 in white-opaque switching is still vague. In this study, we obtain a series of Wor1-interacting proteins using LexA-Wor1 as bait. Among these proteins, function unknown now 30 (Fun30) interacts with Wor1 in vitro and in vivo. Fun30 expression is upregulated in opaque cells at the transcriptional and protein levels. Loss of FUN30 attenuates white-to-opaque switching, while ectopic expression of FUN30 significantly increases white-to-opaque switching in an ATPase activity-dependent manner. Furthermore, FUN30 upregulation is dependent on CO 2; loss of FLO8, a key CO 2-sensing transcriptional regulator, abolishes FUN30 upregulation. Interestingly, deletion of FUN30 affects the WOR1 expression regulation feedback loop. Thus, our results indicate that the chromatin remodeller Fun30 interacts with Wor1 and is required for WOR1 expression and opaque cell formation.
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Affiliation(s)
- Ning Gao
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China
| | - Baodi Dai
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China
| | - Xinyi Nie
- Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
| | - Qun Zhao
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China
| | - Wencheng Zhu
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China.,Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China
| | - Jiangye Chen
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China
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18
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Shi M, Zhao J, Zhang S, Huang W, Li M, Bai X, Zhang W, Zhang K, Chen X, Xiang S. Structural basis for the Rad6 activation by the Bre1 N-terminal domain. eLife 2023; 12:84157. [PMID: 36912886 PMCID: PMC10036116 DOI: 10.7554/elife.84157] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2022] [Accepted: 03/10/2023] [Indexed: 03/14/2023] Open
Abstract
The mono-ubiquitination of the histone protein H2B (H2Bub1) is a highly conserved histone post-translational modification that plays critical roles in many fundamental processes. In yeast, this modification is catalyzed by the conserved Bre1-Rad6 complex. Bre1 contains a unique N-terminal Rad6-binding domain (RBD), how it interacts with Rad6 and contributes to the H2Bub1 catalysis is unclear. Here, we present crystal structure of the Bre1 RBD-Rad6 complex and structure-guided functional studies. Our structure provides a detailed picture of the interaction between the dimeric Bre1 RBD and a single Rad6 molecule. We further found that the interaction stimulates Rad6's enzymatic activity by allosterically increasing its active site accessibility and likely contribute to the H2Bub1 catalysis through additional mechanisms. In line with these important functions, we found that the interaction is crucial for multiple H2Bub1-regulated processes. Our study provides molecular insights into the H2Bub1 catalysis.
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Affiliation(s)
- Meng Shi
- Department of Biochemistry and Molecular Biology, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The province and ministry co-sponsored collaborative innovation center for medical epigenetics, Tianjin Medical University, Tianjin, China
| | - Jiaqi Zhao
- Department of Biochemistry and Molecular Biology, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The province and ministry co-sponsored collaborative innovation center for medical epigenetics, Tianjin Medical University, Tianjin, China
| | - Simin Zhang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, The Institute of Advanced Studies, Wuhan University, Wuhan, China
| | - Wei Huang
- Department of Biochemistry and Molecular Biology, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The province and ministry co-sponsored collaborative innovation center for medical epigenetics, Tianjin Medical University, Tianjin, China
| | - Mengfei Li
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, The Institute of Advanced Studies, Wuhan University, Wuhan, China
| | - Xue Bai
- Department of Biochemistry and Molecular Biology, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The province and ministry co-sponsored collaborative innovation center for medical epigenetics, Tianjin Medical University, Tianjin, China
| | - Wenxue Zhang
- Department of Radiation Oncology, Tianjin Medical University General Hospital, Tianjin, China
| | - Kai Zhang
- Department of Biochemistry and Molecular Biology, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The province and ministry co-sponsored collaborative innovation center for medical epigenetics, Tianjin Medical University, Tianjin, China
| | - Xuefeng Chen
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Centre of Immunology and Metabolism, The Institute of Advanced Studies, Wuhan University, Wuhan, China
| | - Song Xiang
- Department of Biochemistry and Molecular Biology, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The province and ministry co-sponsored collaborative innovation center for medical epigenetics, Tianjin Medical University, Tianjin, China
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19
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Ceppi I, Cannavo E, Bret H, Camarillo R, Vivalda F, Thakur RS, Romero-Franco A, Sartori AA, Huertas P, Guérois R, Cejka P. PLK1 regulates CtIP and DNA2 interplay in long-range DNA end resection. Genes Dev 2023; 37:119-135. [PMID: 36746606 PMCID: PMC10069449 DOI: 10.1101/gad.349981.122] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Accepted: 01/12/2023] [Indexed: 02/08/2023]
Abstract
DNA double-strand break (DSB) repair is initiated by DNA end resection. CtIP acts in short-range resection to stimulate MRE11-RAD50-NBS1 (MRN) to endonucleolytically cleave 5'-terminated DNA to bypass protein blocks. CtIP also promotes the DNA2 helicase-nuclease to accelerate long-range resection downstream from MRN. Here, using AlphaFold2, we identified CtIP-F728E-Y736E as a separation-of-function mutant that is still proficient in conjunction with MRN but is not able to stimulate ssDNA degradation by DNA2. Accordingly, CtIP-F728E-Y736E impairs physical interaction with DNA2. Cellular assays revealed that CtIP-F728E-Y736E cells exhibit reduced DSB-dependent chromatin-bound RPA, impaired long-range resection, and increased sensitivity to DSB-inducing drugs. Previously, CtIP was shown to be targeted by PLK1 to inhibit long-range resection, yet the underlying mechanism was unclear. We show that the DNA2-interacting region in CtIP includes the PLK1 target site at S723. The integrity of S723 in CtIP is necessary for the stimulation of DNA2, and phosphorylation of CtIP by PLK1 in vitro is consequently inhibitory, explaining why PLK1 restricts long-range resection. Our data support a model in which CDK-dependent phosphorylation of CtIP activates resection by MRN in S phase, and PLK1-mediated phosphorylation of CtIP disrupts CtIP stimulation of DNA2 to attenuate long-range resection later at G2/M.
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Affiliation(s)
- Ilaria Ceppi
- Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona 6500, Switzerland
- Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), Zürich 8093, Switzerland
| | - Elda Cannavo
- Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona 6500, Switzerland
| | - Hélène Bret
- Institute for Integrative Biology of the Cell (I2BC), Commissariat à l'Energie Atomique, Centre National de la Recherche Scientifique, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette 91190, France
| | - Rosa Camarillo
- Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Sevilla 41080, Spain
- Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-Consejo Superior de Investigaciones Científicas-Universidad Pablo de Olavide, Sevilla 41092, Spain
| | - Francesca Vivalda
- Institute of Molecular Cancer Research, University of Zürich, Zürich 8057, Switzerland
| | - Roshan Singh Thakur
- Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona 6500, Switzerland
| | - Amador Romero-Franco
- Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Sevilla 41080, Spain
- Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-Consejo Superior de Investigaciones Científicas-Universidad Pablo de Olavide, Sevilla 41092, Spain
| | - Alessandro A Sartori
- Institute of Molecular Cancer Research, University of Zürich, Zürich 8057, Switzerland
| | - Pablo Huertas
- Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Sevilla 41080, Spain
- Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-Consejo Superior de Investigaciones Científicas-Universidad Pablo de Olavide, Sevilla 41092, Spain
| | - Raphaël Guérois
- Institute for Integrative Biology of the Cell (I2BC), Commissariat à l'Energie Atomique, Centre National de la Recherche Scientifique, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette 91190, France
| | - Petr Cejka
- Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona 6500, Switzerland;
- Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), Zürich 8093, Switzerland
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20
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Tóth A, Székvölgyi L, Vellai T. The genome loading model for the origin and maintenance of sex in eukaryotes. Biol Futur 2022; 73:345-357. [PMID: 36534301 DOI: 10.1007/s42977-022-00148-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Accepted: 12/08/2022] [Indexed: 12/23/2022]
Abstract
Understanding why sexual reproduction-which involves syngamy (union of gametes) and meiosis-emerged and how it has subsisted for millions of years remains a fundamental problem in biology. Considered as the essence of sex, meiotic recombination is initiated by a DNA double-strand break (DSB) that forms on one of the pairing homologous chromosomes. This DNA lesion is subsequently repaired by gene conversion, the non-reciprocal transfer of genetic information from the intact homolog. A major issue is which of the pairing homologs undergoes DSB formation. Accumulating evidence shows that chromosomal sites where the pairing homologs locally differ in size, i.e., are heterozygous for an insertion or deletion, often display disparity in gene conversion. Biased conversion tends to duplicate insertions and lose deletions. This suggests that DSB is preferentially formed on the "shorter" homologous region, which thereby acts as the recipient for DNA transfer. Thus, sex primarily functions as a genome (re)loading mechanism. It ensures the restoration of formerly lost DNA sequences (deletions) and allows the efficient copying and, mainly in eukaryotes, subsequent spreading of newly emerged sequences (insertions) arising initially in an individual genome, even if they confer no advantage to the host. In this way, sex simultaneously repairs deletions and increases genetic variability underlying adaptation. The model explains a remarkable increase in DNA content during the evolution of eukaryotic genomes.
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Affiliation(s)
- András Tóth
- Department of Genetics, Eötvös Loránd University, Pázmány Péter Stny. 1/C, Budapest, 1117, Hungary
| | - Lóránt Székvölgyi
- MTA-DE Momentum Genome Architecture and Recombination Research Group, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, 4032, Hungary
| | - Tibor Vellai
- Department of Genetics, Eötvös Loránd University, Pázmány Péter Stny. 1/C, Budapest, 1117, Hungary.
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21
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Kloeber JA, Lou Z. Critical DNA damaging pathways in tumorigenesis. Semin Cancer Biol 2022; 85:164-184. [PMID: 33905873 PMCID: PMC8542061 DOI: 10.1016/j.semcancer.2021.04.012] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2021] [Revised: 04/13/2021] [Accepted: 04/15/2021] [Indexed: 12/22/2022]
Abstract
The acquisition of DNA damage is an early driving event in tumorigenesis. Premalignant lesions show activated DNA damage responses and inactivation of DNA damage checkpoints promotes malignant transformation. However, DNA damage is also a targetable vulnerability in cancer cells. This requires a detailed understanding of the cellular and molecular mechanisms governing DNA integrity. Here, we review current work on DNA damage in tumorigenesis. We discuss DNA double strand break repair, how repair pathways contribute to tumorigenesis, and how double strand breaks are linked to the tumor microenvironment. Next, we discuss the role of oncogenes in promoting DNA damage through replication stress. Finally, we discuss our current understanding on DNA damage in micronuclei and discuss therapies targeting these DNA damage pathways.
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Affiliation(s)
- Jake A Kloeber
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA; Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, 55905, USA; Mayo Clinic Medical Scientist Training Program, Mayo Clinic, Rochester, MN, 55905, USA
| | - Zhenkun Lou
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA.
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22
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Chen BR, Sleckman BP. The regulation of DNA end resection by chromatin response to DNA double strand breaks. Front Cell Dev Biol 2022; 10:932633. [PMID: 35912102 PMCID: PMC9335370 DOI: 10.3389/fcell.2022.932633] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2022] [Accepted: 06/29/2022] [Indexed: 11/18/2022] Open
Abstract
DNA double-strand breaks (DSBs) constantly arise upon exposure to genotoxic agents and during physiological processes. The timely repair of DSBs is important for not only the completion of the cellular functions involving DSBs as intermediates, but also the maintenance of genome stability. There are two major pathways dedicated to DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). The decision of deploying HR or NHEJ to repair DSBs largely depends on the structures of broken DNA ends. DNA ends resected to generate extensive single-strand DNA (ssDNA) overhangs are repaired by HR, while those remaining blunt or minimally processed can be repaired by NHEJ. As the generation and repair of DSB occurs within the context of chromatin, the resection of broken DNA ends is also profoundly affected by the state of chromatin flanking DSBs. Here we review how DNA end resection can be regulated by histone modifications, chromatin remodeling, and the presence of ssDNA structure through altering the accessibility to chromatin and the activity of pro- and anti-resection proteins.
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Affiliation(s)
- Bo-Ruei Chen
- Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States
- O’Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, United States
- *Correspondence: Bo-Ruei Chen,
| | - Barry P. Sleckman
- Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States
- O’Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, United States
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23
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Chen Z, Tyler JK. The Chromatin Landscape Channels DNA Double-Strand Breaks to Distinct Repair Pathways. Front Cell Dev Biol 2022; 10:909696. [PMID: 35757003 PMCID: PMC9213757 DOI: 10.3389/fcell.2022.909696] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Accepted: 05/17/2022] [Indexed: 12/24/2022] Open
Abstract
DNA double-strand breaks (DSBs), the most deleterious DNA lesions, are primarily repaired by two pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ), the choice of which is largely dependent on cell cycle phase and the local chromatin landscape. Recent studies have revealed that post-translational modifications on histones play pivotal roles in regulating DSB repair pathways including repair pathway choice. In this review, we present our current understanding of how these DSB repair pathways are employed in various chromatin landscapes to safeguard genomic integrity. We place an emphasis on the impact of different histone post-translational modifications, characteristic of euchromatin or heterochromatin regions, on DSB repair pathway choice. We discuss the potential roles of damage-induced chromatin modifications in the maintenance of genome and epigenome integrity. Finally, we discuss how RNA transcripts from the vicinity of DSBs at actively transcribed regions also regulate DSB repair pathway choice.
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Affiliation(s)
- Zulong Chen
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York City, NY, United States
| | - Jessica K Tyler
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York City, NY, United States
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24
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Fun30 and Rtt109 Mediate Epigenetic Regulation of the DNA Damage Response Pathway in C. albicans. J Fungi (Basel) 2022; 8:jof8060559. [PMID: 35736042 PMCID: PMC9225650 DOI: 10.3390/jof8060559] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2022] [Revised: 03/27/2022] [Accepted: 03/29/2022] [Indexed: 01/27/2023] Open
Abstract
Fun30, an ATP-dependent chromatin remodeler from S. cerevisiae, is known to mediate both regulation of gene expression as well as DNA damage response/repair. The Fun30 from C. albicans has not yet been elucidated. We show that C. albicans Fun30 is functionally homologous to both S. cerevisiae Fun30 and human SMARCAD1. Further, C. albicans Fun30 can mediate double-strand break end resection as well as regulate gene expression. This protein regulates transcription of RTT109, TEL1, MEC1, and SNF2-genes that encode for proteins involved in DNA damage response and repair pathways. The regulation mediated by C. albicans Fun30 is dependent on its ATPase activity. The expression of FUN30, in turn, is regulated by histone H3K56 acetylation catalyzed by Rtt109 and encoded by RTT109. The RTT109Hz/FUN30Hz mutant strain shows sensitivity to oxidative stress and resistance to MMS as compared to the wild-type strain. Quantitative PCR showed that the sensitivity to oxidative stress results from downregulation of MEC1, RAD9, MRC1, and RAD5 expression; ChIP experiments showed that Fun30 but not H3K56ac regulates the expression of these genes in response to oxidative stress. In contrast, upon treatment with MMS, the expression of RAD9 is upregulated, which is modulated by both Fun30 and H3K56 acetylation. Thus, Fun30 and H3K56 acetylation mediate the response to genotoxic agents in C. albicans by regulating the expression of DNA damage response and repair pathway genes.
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25
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Scherzer M, Giordano F, Ferran MS, Ström L. Recruitment of Scc2/4 to double-strand breaks depends on γH2A and DNA end resection. Life Sci Alliance 2022; 5:e202101244. [PMID: 35086935 PMCID: PMC8807874 DOI: 10.26508/lsa.202101244] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 01/13/2022] [Accepted: 01/14/2022] [Indexed: 12/15/2022] Open
Abstract
Homologous recombination enables cells to overcome the threat of DNA double-strand breaks (DSBs), allowing for repair without the loss of genetic information. Central to the homologous recombination repair process is the de novo loading of cohesin around a DSB by its loader complex Scc2/4. Although cohesin's DSB accumulation has been explored in numerous studies, the prerequisites for Scc2/4 recruitment during the repair process are still elusive. To address this question, we combine chromatin immunoprecipitation-qPCR with a site-specific DSB in vivo, in Saccharomyces cerevisiae We find that Scc2 DSB recruitment relies on γH2A and Tel1, but as opposed to cohesin, not on Mec1. We further show that the binding of Scc2, which emanates from the break site, depends on and coincides with DNA end resection. Absence of chromatin remodeling at the DSB affects Scc2 binding and DNA end resection to a comparable degree, further indicating the latter to be a major driver for Scc2 recruitment. Our results shed light on the intricate DSB repair cascade leading to the recruitment of Scc2/4 and subsequent loading of cohesin.
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Affiliation(s)
- Martin Scherzer
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Fosco Giordano
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Maria Solé Ferran
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Lena Ström
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
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26
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Modulating DNA Repair Pathways to Diversify Genomic Alterations in Saccharomyces cerevisiae. Microbiol Spectr 2022; 10:e0232621. [PMID: 35352941 PMCID: PMC9045378 DOI: 10.1128/spectrum.02326-21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Nuclease based genome editing systems have emerged as powerful tools to drive genomic alterations and enhance genome evolution via precise engineering in the various human and microbial cells. However, error-prone DNA repair has not been well studied previously to generate diverse genomic alterations and novel phenotypes. Here, we systematically investigated the potential interplay between DNA double strand break (DSB) repair and genome editing tools, and found that modulating the DSB end resection proteins could significantly improve mutational efficiency and diversity without exogenous DNA template in yeast. Deleting SAE2, EXO1, or FUN30, or overexpressing MRE11-H125N (nuclease-dead allele of MRE11), for DSB end resection markedly increased the efficiency of CRISPR/SpCas9 (more than 22-fold) and CRISPR/AsCpf1 (more than 30-fold)-induced mutagenesis. Deleting SAE2 or overexpressing MRE11-H125N substantially diversified CRISPR/SpCas9 or AsCpf1-induced mutation 2–3-fold at URA3 locus, and 3–5-fold at ADE2 locus. Thus, the error-prone DNA repair protein was employed to develop a novel mutagenic genome editing (mGE) strategy, which can increase the mutation numbers and effectively improve the ethanol/glycerol ratio of Saccharomyces cerevisiae through modulating the expression of FPS1 and GPD1. This study highlighted the feasibility of potentially reshaping the capability of genome editing by regulating the different DSB repair proteins and can thus expand the application of genome editing in diversifying gene expression and enhancing genome evolution. IMPORTANCE Most of the published papers about nuclease-assisted genome editing focused on precision engineering in human cells. However, the topic of inducing mutagenesis via error-prone repair has often been ignored in yeast. In this study, we reported that perturbing DNA repair, especially modifications of the various DSB end resection-related proteins, could greatly improve the mutational efficiency and diversity, and thus functionally reshape the capability of the different genome editing tools without requiring an exogenous DNA template in yeast. Specifically, mutagenic genome editing (mGE) was developed based on CRISPR/AsCpf1 and MRE11-H125N overexpression, and used to generate promoters of different strengths more efficiently. Thus, this work provides a novel method to diversify gene expression and enhance genome evolution.
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27
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Kang H, Liu Y, Fan T, Ma J, Wu D, Heitz T, Shen WH, Zhu Y. Arabidopsis CHROMATIN REMODELING 19 acts as a transcriptional repressor and contributes to plant pathogen resistance. THE PLANT CELL 2022; 34:1100-1116. [PMID: 34954802 PMCID: PMC8894922 DOI: 10.1093/plcell/koab318] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/01/2021] [Accepted: 12/21/2021] [Indexed: 06/14/2023]
Abstract
Chromatin remodelers act in an ATP-dependent manner to modulate chromatin structure and thus genome function. Here, we report that the Arabidopsis (Arabidopsis thaliana) remodeler CHROMATIN REMODELING19 (CHR19) is enriched in gene body regions, and its depletion causes massive changes in nucleosome position and occupancy in the genome. Consistent with these changes, an in vitro assay verified that CHR19 can utilize ATP to slide nucleosomes. A variety of inducible genes, including several important genes in the salicylic acid (SA) and jasmonic acid (JA) pathways, were transcriptionally upregulated in the chr19 mutant under normal growth conditions, indicative of a role of CHR19 in transcriptional repression. In addition, the chr19 mutation triggered higher susceptibility to the JA pathway-defended necrotrophic fungal pathogen Botrytis cinerea, but did not affect the growth of the SA pathway-defended hemibiotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Expression of CHR19 was tissue-specific and inhibited specifically by SA treatment. Such inhibition significantly decreased the local chromatin enrichment of CHR19 at the associated SA pathway genes, which resulted in their full activation upon SA treatment. Overall, our findings clarify CHR19 to be a novel regulator acting at the chromatin level to impact the transcription of genes underlying plant resistance to different pathogens.
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Affiliation(s)
- Huijia Kang
- Department of Biochemistry, Institute of Plant Biology, School of Life
Sciences, State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for
Genetics and Development, Fudan University, Shanghai 200438, China
- Institut de Biologie Moléculaire des Plantes, CNRS, Université de
Strasbourg, Strasbourg Cedex 67084, France
| | - Yuhao Liu
- National Cancer Center/National Clinical Research Center for Cancer/Cancer
Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union
Medical College, Shenzhen 518116, China; Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing, 100021,
China
| | - Tianyi Fan
- Department of Biochemistry, Institute of Plant Biology, School of Life
Sciences, State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for
Genetics and Development, Fudan University, Shanghai 200438, China
| | - Jing Ma
- Department of Biochemistry, Institute of Plant Biology, School of Life
Sciences, State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for
Genetics and Development, Fudan University, Shanghai 200438, China
| | - Di Wu
- Department of Biochemistry, Institute of Plant Biology, School of Life
Sciences, State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for
Genetics and Development, Fudan University, Shanghai 200438, China
| | - Thierry Heitz
- Institut de Biologie Moléculaire des Plantes, CNRS, Université de
Strasbourg, Strasbourg Cedex 67084, France
| | - Wen-Hui Shen
- Institut de Biologie Moléculaire des Plantes, CNRS, Université de
Strasbourg, Strasbourg Cedex 67084, France
| | - Yan Zhu
- Department of Biochemistry, Institute of Plant Biology, School of Life
Sciences, State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for
Genetics and Development, Fudan University, Shanghai 200438, China
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28
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Karl LA, Peritore M, Galanti L, Pfander B. DNA Double Strand Break Repair and Its Control by Nucleosome Remodeling. Front Genet 2022; 12:821543. [PMID: 35096025 PMCID: PMC8790285 DOI: 10.3389/fgene.2021.821543] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2021] [Accepted: 12/23/2021] [Indexed: 12/12/2022] Open
Abstract
DNA double strand breaks (DSBs) are repaired in eukaryotes by one of several cellular mechanisms. The decision-making process controlling DSB repair takes place at the step of DNA end resection, the nucleolytic processing of DNA ends, which generates single-stranded DNA overhangs. Dependent on the length of the overhang, a corresponding DSB repair mechanism is engaged. Interestingly, nucleosomes-the fundamental unit of chromatin-influence the activity of resection nucleases and nucleosome remodelers have emerged as key regulators of DSB repair. Nucleosome remodelers share a common enzymatic mechanism, but for global genome organization specific remodelers have been shown to exert distinct activities. Specifically, different remodelers have been found to slide and evict, position or edit nucleosomes. It is an open question whether the same remodelers exert the same function also in the context of DSBs. Here, we will review recent advances in our understanding of nucleosome remodelers at DSBs: to what extent nucleosome sliding, eviction, positioning and editing can be observed at DSBs and how these activities affect the DSB repair decision.
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Affiliation(s)
- Leonhard Andreas Karl
- Resarch Group DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Martina Peritore
- Resarch Group DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Lorenzo Galanti
- Resarch Group DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Boris Pfander
- Resarch Group DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
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29
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Oh JM, Myung K. Crosstalk between different DNA repair pathways for DNA double strand break repairs. MUTATION RESEARCH. GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2022; 873:503438. [PMID: 35094810 DOI: 10.1016/j.mrgentox.2021.503438] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/09/2021] [Revised: 11/09/2021] [Accepted: 12/14/2021] [Indexed: 11/28/2022]
Abstract
DNA double strand breaks (DSBs) are the most threatening type of DNA lesions and must be repaired properly in order to inhibit severe diseases and cell death. There are four major repair pathways for DSBs: non-homologous end joining (NHEJ), homologous recombination (HR), single strand annealing (SSA) and alternative end joining (alt-EJ). Cells choose repair pathway depending on the cell cycle phase and the length of 3' end of the DNA when DSBs are generated. Blunt and short regions of the 5' or 3' overhang DNA are repaired by NHEJ, which uses direct ligation or limited resection processing of the broken DNA end. In contrast, HR, SSA and alt-EJ use the resected DNA generated by the MRN (MRE11-RAD50-NBS1) complex and C-terminal binding protein interacting protein (CtIP) activated during the S and G2 phases. Here, we review recent findings on each repair pathway and the choice of repair mechanism and highlight the role of mismatch repair (MMR) protein in HR.
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Affiliation(s)
- Jung-Min Oh
- Department of Oral Biochemistry, Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea.
| | - Kyungjae Myung
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea; Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea.
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30
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Pandita TK, Hunt CR, Singh V, Adhikary S, Pandita S, Roy S, Ramos K, Das C. Role of the Histone Acetyl Transferase MOF and the Histone Deacetylase Sirtuins in Regulation of H4K16ac During DNA Damage Repair and Metabolic Programming: Implications in Cancer and Aging. Subcell Biochem 2022; 100:115-141. [PMID: 36301493 DOI: 10.1007/978-3-031-07634-3_4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/16/2023]
Abstract
The accurate repair of genomic damage mediated by ionizing radiation (IR), chemo- or radiomimetic drugs, or other exogenous agents, is necessary for maintenance of genome integrity, preservation of cellular viability and prevention of oncogenic transformation. Eukaryotes have conserved mechanisms designed to perceive and repair the damaged DNA quite efficiently. Among the different types of DNA damage, double strand breaks (DSB) are the most detrimental. The cellular DNA DSB response is a hierarchical signaling network that integrates damage sensing and repair with chromatin structural changes that involve a range of pre-existing and induced covalent modifications. Recent studies have revealed that pre-existing histone modifications are important contributors within this signaling/repair network. This chapter discusses the role of a critical histone acetyl transferase (HAT) known as MOF (males absent on the first) and the histone deacetylases (HDACs) Sirtuins on histone H4K16 acetylation (H4K16ac) and DNA damage repair. We also discuss the role of this important histone modification in light of metabolic rewiring and its role in regulating human pathophysiologic states.
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Affiliation(s)
- Tej K Pandita
- The Houston Methodist Research Institute, Houston, TX, USA.
- Department of Cellular and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.
- Center for Genomics and Precision Medicine, Texas A&M College of Medicine, Houston, TX, USA.
| | - Clayton R Hunt
- The Houston Methodist Research Institute, Houston, TX, USA
| | - Vipin Singh
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India
- Homi Bhaba National Institute, Mumbai, India
| | - Santanu Adhikary
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India
- Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India
| | - Shruti Pandita
- Department of Internal Medicine, Division of Hematology, Oncology and Cellular Therapy, Saint Louis University School of Medicine, St. Louis, MO, USA
| | - Siddhartha Roy
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India
| | - Kenneth Ramos
- Center for Genomics and Precision Medicine, Texas A&M College of Medicine, Houston, TX, USA
| | - Chandrima Das
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, India
- Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India
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31
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De Falco M, De Felice M. Take a Break to Repair: A Dip in the World of Double-Strand Break Repair Mechanisms Pointing the Gaze on Archaea. Int J Mol Sci 2021; 22:ijms222413296. [PMID: 34948099 PMCID: PMC8708640 DOI: 10.3390/ijms222413296] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Revised: 12/06/2021] [Accepted: 12/07/2021] [Indexed: 12/24/2022] Open
Abstract
All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases.
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Abstract
DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5'→3' nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.
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Affiliation(s)
- Petr Cejka
- Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), 6500 Bellinzona, Switzerland; .,Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH) Zürich, 8093 Zürich, Switzerland
| | - Lorraine S Symington
- Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA; .,Department of Genetics & Development, Columbia University Irving Medical Center, New York, NY 10032, USA
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33
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DNA Damage-Induced Phosphorylation of Histone H2A at Serine 15 Is Linked to DNA End Resection. Mol Cell Biol 2021; 41:e0005621. [PMID: 34570618 DOI: 10.1128/mcb.00056-21] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
The repair of DNA double-strand breaks (DSBs) occurs in chromatin, and several histone posttranslational modifications have been implicated in the process. Modifications of the histone H2A N-terminal tail have also been linked to DNA damage response, through acetylation or ubiquitination of lysine residues that regulate repair pathway choice. Here, we characterize a new DNA damage-induced phosphorylation on chromatin, at serine 15 of H2A in yeast. We show that this SQ motif functions independently of the classical S129 C-terminal site (γ-H2A) and that mutant-mimicking constitutive phosphorylation increases cell sensitivity to DNA damage. H2AS129ph is induced by Tel1ATM and Mec1ATR, and the loss of Lcd1ATRIP or Mec1 signaling decreases γ-H2A spreading distal to the DSB. In contrast, H2AS15ph is completely dependent on Lcd1ATRIP, indicating that this modification only happens when end resection is engaged. This is supported by an increase in replication protein A (RPA) and a decrease in DNA signal near the DSB in H2A-S15E phosphomimic mutants, indicating higher resection. In mammals, this serine is replaced by a lysine (H2AK15) which undergoes an acetyl-monoubiquityl switch to regulate binding of 53BP1 and resection. This regulation seems functionally conserved with budding yeast H2AS15 and 53BP1-homolog Rad9, using different posttranslational modifications between organisms but achieving the same function.
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34
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Han H, Jiang G, Kumari R, Silic MR, Owens JL, Hu C, Mittal SK, Zhang G. Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish. Genes Chromosomes Cancer 2021; 60:743-761. [PMID: 34296799 PMCID: PMC9585957 DOI: 10.1002/gcc.22983] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2020] [Revised: 07/02/2021] [Accepted: 07/05/2021] [Indexed: 11/21/2022] Open
Abstract
Malignant peripheral nerve sheath tumors (MPNSTs) are a type of sarcoma that generally originates from Schwann cells. The prognosis for this type of malignancy is relatively poor due to complicated genetic alterations and the lack of specific targeted therapy. Chromosome fragment 4q22-23 is frequently deleted in MPNSTs and other human tumors, suggesting tumor suppressor genes may reside in this region. Here, we provide evidence that SMARCAD1, a known chromatin remodeler, is a novel tumor suppressor gene located in 4q22-23. We identified two human homologous smarcad1 genes (smarcad1a and smarcad1b) in zebrafish, and both genes share overlapping expression patterns during embryonic development. We demonstrated that two smarcad1a loss-of-function mutants, sa1299 and p403, can accelerate MPNST tumorigenesis in the tp53 mutant background, suggesting smarcad1a is a bona fide tumor suppressor gene for MPNSTs. Moreover, we found that DNA double-strand break (DSB) repair might be compromised in both mutants compared to wildtype zebrafish, as indicated by pH2AX, a DNA DSB marker. In addition, both SMARCAD1 gene knockdown and overexpression in human cells were able to inhibit tumor growth and displayed similar DSB repair responses, suggesting proper SMARCAD1 gene expression level or gene dosage is critical for cell growth. Given that mutations of SMARCAD1 sensitize cells to poly ADP ribose polymerase inhibitors in yeast and the human U2OS osteosarcoma cell line, the identification of SMARCAD1 as a novel tumor suppressor gene might contribute to the development of new cancer therapies for MPNSTs.
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Affiliation(s)
- Han Han
- Department of Comparative PathobiologyPurdue UniversityWest LafayetteIndianaUSA
| | - Guangzhen Jiang
- Department of Comparative PathobiologyPurdue UniversityWest LafayetteIndianaUSA
- Present address:
College of Animal Science and TechnologyNanjing Agricultural UniversityNanjingChina
| | - Rashmi Kumari
- Department of Comparative PathobiologyPurdue UniversityWest LafayetteIndianaUSA
| | - Martin R. Silic
- Department of Comparative PathobiologyPurdue UniversityWest LafayetteIndianaUSA
| | - Jake L. Owens
- Department of Medicinal Chemistry and Molecular PharmacologyPurdue UniversityWest LafayetteIndianaUSA
| | - Chang‐Deng Hu
- Department of Medicinal Chemistry and Molecular PharmacologyPurdue UniversityWest LafayetteIndianaUSA
- Purdue University Center for Cancer ResearchPurdue UniversityWest LafayetteIndianaUSA
| | - Suresh K. Mittal
- Department of Comparative PathobiologyPurdue UniversityWest LafayetteIndianaUSA
- Purdue University Center for Cancer ResearchPurdue UniversityWest LafayetteIndianaUSA
- Purdue Institute for Inflammation, Immunology and Infectious Disease (PI4D)Purdue UniversityWest LafayetteIndianaUSA
| | - GuangJun Zhang
- Department of Comparative PathobiologyPurdue UniversityWest LafayetteIndianaUSA
- Purdue University Center for Cancer ResearchPurdue UniversityWest LafayetteIndianaUSA
- Purdue Institute for Inflammation, Immunology and Infectious Disease (PI4D)Purdue UniversityWest LafayetteIndianaUSA
- Purdue Institute for Integrative Neuroscience (PIIN)Purdue UniversityWest LafayetteIndianaUSA
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35
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Mohan C, Das C, Tyler J. Histone and Chromatin Dynamics Facilitating DNA repair. DNA Repair (Amst) 2021; 107:103183. [PMID: 34419698 PMCID: PMC9733910 DOI: 10.1016/j.dnarep.2021.103183] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Revised: 07/15/2021] [Accepted: 07/16/2021] [Indexed: 12/13/2022]
Abstract
Our nuclear genomes are complexed with histone proteins to form nucleosomes, the repeating units of chromatin which function to package and limit unscheduled access to the genome. In response to helix-distorting DNA lesions and DNA double-strand breaks, chromatin is disassembled around the DNA lesion to facilitate DNA repair and it is reassembled after repair is complete to reestablish the epigenetic landscape and regulating access to the genome. DNA damage also triggers decondensation of the local chromatin structure, incorporation of histone variants and dramatic transient increases in chromatin mobility to facilitate the homology search during homologous recombination. Here we review the current state of knowledge of these changes in histone and chromatin dynamics in response to DNA damage, the molecular mechanisms mediating these dynamics, as well as their functional contributions to the maintenance of genome integrity to prevent human diseases including cancer.
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Affiliation(s)
- Chitra Mohan
- Department of Pathology and Laboratory Medicine, 1300 York Avenue, New York, NY, 10065, USA
| | - Chandrima Das
- Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata, 700064, India
| | - Jessica Tyler
- Department of Pathology and Laboratory Medicine, 1300 York Avenue, New York, NY, 10065, USA.
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36
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Chakraborty U, Shen ZJ, Tyler J. Chaperoning histones at the DNA repair dance. DNA Repair (Amst) 2021; 108:103240. [PMID: 34687987 DOI: 10.1016/j.dnarep.2021.103240] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2021] [Revised: 09/28/2021] [Accepted: 10/03/2021] [Indexed: 12/15/2022]
Abstract
Unlike all other biological molecules that are degraded and replaced if damaged, DNA must be repaired as chromosomes cannot be replaced. Indeed, DNA endures a wide variety of structural damage that need to be repaired accurately to maintain genomic stability and proper functioning of cells and to prevent mutation leading to disease. Given that the genome is packaged into chromatin within eukaryotic cells, it has become increasingly evident that the chromatin context of DNA both facilitates and regulates DNA repair processes. In this review, we discuss mechanisms involved in removal of histones (chromatin disassembly) from around DNA lesions, by histone chaperones and chromatin remodelers, that promotes accessibility of the DNA repair machinery. We also elaborate on how the deposition of core histones and specific histone variants onto DNA (chromatin assembly) during DNA repair promotes repair processes, the role of histone post translational modifications in these processes and how chromatin structure is reestablished after DNA repair is complete.
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Affiliation(s)
- Ujani Chakraborty
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, 1300 York Avenue, New York, NY 10065, USA
| | - Zih-Jie Shen
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, 1300 York Avenue, New York, NY 10065, USA
| | - Jessica Tyler
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, 1300 York Avenue, New York, NY 10065, USA.
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37
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Markert J, Zhou K, Luger K. SMARCAD1 is an ATP-dependent histone octamer exchange factor with de novo nucleosome assembly activity. SCIENCE ADVANCES 2021; 7:eabk2380. [PMID: 34652950 PMCID: PMC8519567 DOI: 10.1126/sciadv.abk2380] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/13/2023]
Abstract
The adenosine 5′-triphosphate (ATP)–dependent chromatin remodeler SMARCAD1 acts on nucleosomes during DNA replication, repair, and transcription, but despite its implication in disease, information on its function and biochemical activities is scarce. Chromatin remodelers use the energy of ATP hydrolysis to slide nucleosomes, evict histones, or exchange histone variants. Here, we show that SMARCAD1 transfers the entire histone octamer from one DNA segment to another in an ATP-dependent manner but is also capable of de novo nucleosome assembly from histone octamer because of its ability to simultaneously bind all histones. We present a low-resolution cryo–electron microscopy structure of SMARCAD1 in complex with a nucleosome and show that the adenosine triphosphatase domains engage their substrate unlike any other chromatin remodeler. Our biochemical and structural data provide mechanistic insights into SMARCAD1-induced nucleosome disassembly and reassembly.
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Affiliation(s)
- Jonathan Markert
- Department of Biochemistry, University of Colorado Boulder, Boulder, CO 80303, USA
| | - Keda Zhou
- Department of Biochemistry, University of Colorado Boulder, Boulder, CO 80303, USA
| | - Karolin Luger
- Department of Biochemistry, University of Colorado Boulder, Boulder, CO 80303, USA
- Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
- Corresponding author.
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38
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The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks. PLoS Genet 2021; 17:e1009807. [PMID: 34520455 PMCID: PMC8462745 DOI: 10.1371/journal.pgen.1009807] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2021] [Revised: 09/24/2021] [Accepted: 09/06/2021] [Indexed: 12/31/2022] Open
Abstract
Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5’-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities. DNA double strand breaks (DSBs) are among the most severe types of damage occurring in the genome because their faulty repair can result in chromosome instability, commonly associated with carcinogenesis. Efficient and accurate repair of DSBs relies on several proteins required to process them. However, eukaryotic genomes are compacted into chromatin, which restricts the access to DNA of the enzymes devoted to repair DNA DSBs. To overcome this natural barrier, eukaryotes have evolved chromatin remodeling enzymes that use energy derived from ATP hydrolysis to modulate chromatin structure. Here, we examine the role in DSB repair of the ATP-dependent chromatin remodeler Chd1, which is frequently mutated in prostate cancer. We find that Chd1 is important to repair DNA DSBs by homologous recombination (HR) because it promotes the association with a damaged site of the MRX complex and Exo1, which are necessary to initiate HR. This Chd1 function requires its ATPase activity, suggesting that Chd1 increases the accessibility to chromatin to initiate repair of DNA lesions.
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39
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Guha S, Bhaumik SR. Transcription-coupled DNA double-strand break repair. DNA Repair (Amst) 2021; 109:103211. [PMID: 34883263 DOI: 10.1016/j.dnarep.2021.103211] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Revised: 08/11/2021] [Accepted: 08/11/2021] [Indexed: 12/20/2022]
Abstract
The genomic DNA is constantly under attack by cellular and/or environmental factors. Fortunately, the cell is armed to safeguard its genome by various mechanisms such as nucleotide excision, base excision, mismatch and DNA double-strand break repairs. While these processes maintain the integrity of the genome throughout, DNA repair occurs preferentially faster at the transcriptionally active genes. Such transcription-coupled repair phenomenon plays important roles to maintain active genome integrity, failure of which would interfere with transcription, leading to an altered gene expression (and hence cellular pathologies/diseases). Among the various DNA damages, DNA double-strand breaks are quite toxic to the cells. If DNA double-strand break occurs at the active gene, it would interfere with transcription/gene expression, thus threatening cellular viability. Such DNA double-strand breaks are found to be repaired faster at the active gene in comparison to its inactive state or the inactive gene, thus supporting the existence of a new phenomenon of transcription-coupled DNA double-strand break repair. Here, we describe the advances of this repair process.
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Affiliation(s)
- Shalini Guha
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL, 62901, USA
| | - Sukesh R Bhaumik
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL, 62901, USA.
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40
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Peng H, Zhang S, Peng Y, Zhu S, Zhao X, Zhao X, Yang S, Liu G, Dong Y, Gan X, Li Q, Zhang X, Pei H, Chen X. Yeast Bromodomain Factor 1 and Its Human Homolog TAF1 Play Conserved Roles in Promoting Homologous Recombination. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2021; 8:e2100753. [PMID: 34056863 PMCID: PMC8336524 DOI: 10.1002/advs.202100753] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/23/2021] [Revised: 04/27/2021] [Indexed: 05/12/2023]
Abstract
Histone acetylation is a key histone post-translational modification that shapes chromatin structure, dynamics, and function. Bromodomain (BRD) proteins, the readers of acetyl-lysines, are located in the center of the histone acetylation-signaling network. How they regulate DNA repair and genome stability remains poorly understood. Here, a conserved function of the yeast Bromodomain Factor 1 (Bdf1) and its human counterpart TAF1 is reported in promoting DNA double-stranded break repair by homologous recombination (HR). Depletion of either yeast BDF1 or human TAF1, or disruption of their BRDs impairs DNA end resection, Replication Protein A (RPA) and Rad51 loading, and HR repair, causing genome instability and hypersensitivity to DNA damage. Mechanistically, it is shown that Bdf1 preferentially binds the DNA damage-induced histone H4 acetylation (H4Ac) via the BRD motifs, leading to its chromatin recruitment. Meanwhile, Bdf1 physically interacts with RPA, and this interaction facilitates RPA loading in the chromatin context and the subsequent HR repair. Similarly, TAF1 also interacts with H4Ac or RPA. Thus, Bdf1 and TAF1 appear to share a conserved mechanism in linking the HR repair to chromatin acetylation in preserving genome integrity.
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Affiliation(s)
- Haoyang Peng
- Hubei Key Laboratory of Cell HomeostasisCollege of Life Sciences and the Institute for Advanced StudiesWuhan UniversityWuhan430072China
| | - Simin Zhang
- Hubei Key Laboratory of Cell HomeostasisCollege of Life Sciences and the Institute for Advanced StudiesWuhan UniversityWuhan430072China
| | - Yihan Peng
- Department of Biochemistry and Molecular MedicineGeorge Washington University School of Medicine and Health ScienceWashingtonDC20037USA
| | - Shuangyi Zhu
- Hubei Key Laboratory of Cell HomeostasisCollege of Life Sciences and the Institute for Advanced StudiesWuhan UniversityWuhan430072China
| | - Xin Zhao
- Hubei Key Laboratory of Cell HomeostasisCollege of Life Sciences and the Institute for Advanced StudiesWuhan UniversityWuhan430072China
| | - Xiaocong Zhao
- Hubei Key Laboratory of Cell HomeostasisCollege of Life Sciences and the Institute for Advanced StudiesWuhan UniversityWuhan430072China
| | - Shuangshuang Yang
- State Key Laboratory of Protein and Plant Gene ResearchSchool of Life Sciences and Peking‐Tsinghua Center for Life SciencesPeking UniversityBeijing100871China
| | - Guangxue Liu
- Hubei Key Laboratory of Cell HomeostasisCollege of Life Sciences and the Institute for Advanced StudiesWuhan UniversityWuhan430072China
| | - Yang Dong
- Hubei Key Laboratory of Cell HomeostasisCollege of Life Sciences and the Institute for Advanced StudiesWuhan UniversityWuhan430072China
| | - Xiaoli Gan
- Hubei Key Laboratory of Cell HomeostasisCollege of Life Sciences and the Institute for Advanced StudiesWuhan UniversityWuhan430072China
| | - Qing Li
- State Key Laboratory of Protein and Plant Gene ResearchSchool of Life Sciences and Peking‐Tsinghua Center for Life SciencesPeking UniversityBeijing100871China
| | - Xinghua Zhang
- Hubei Key Laboratory of Cell HomeostasisCollege of Life Sciences and the Institute for Advanced StudiesWuhan UniversityWuhan430072China
| | - Huadong Pei
- Department of Biochemistry and Molecular MedicineGeorge Washington University School of Medicine and Health ScienceWashingtonDC20037USA
| | - Xuefeng Chen
- Hubei Key Laboratory of Cell HomeostasisCollege of Life Sciences and the Institute for Advanced StudiesWuhan UniversityWuhan430072China
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41
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DNA end resection during homologous recombination. Curr Opin Genet Dev 2021; 71:99-105. [PMID: 34329854 DOI: 10.1016/j.gde.2021.07.004] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 07/03/2021] [Accepted: 07/05/2021] [Indexed: 12/15/2022]
Abstract
Exposure to environmental mutagens but also cell-endogenous processes can create DNA double-strand breaks (DSBs) in a cell's genome. DSBs need to be repaired accurately and timely to ensure genomic integrity and cell survival. One major DSB repair mechanism, called homologous recombination, relies on the nucleolytic degradation of the 5'-terminated strands in a process termed end resection. Here, we review new insights into end resection with a focus on the mechanistic interplay of the nucleases, helicases, and accessory factors involved.
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42
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Cheng X, Côté V, Côté J. NuA4 and SAGA acetyltransferase complexes cooperate for repair of DNA breaks by homologous recombination. PLoS Genet 2021; 17:e1009459. [PMID: 34228704 PMCID: PMC8284799 DOI: 10.1371/journal.pgen.1009459] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 07/16/2021] [Accepted: 06/21/2021] [Indexed: 12/30/2022] Open
Abstract
Chromatin modifying complexes play important yet not fully defined roles in DNA repair processes. The essential NuA4 histone acetyltransferase (HAT) complex is recruited to double-strand break (DSB) sites and spreads along with DNA end resection. As predicted, NuA4 acetylates surrounding nucleosomes upon DSB induction and defects in its activity correlate with altered DNA end resection and Rad51 recombinase recruitment. Importantly, we show that NuA4 is also recruited to the donor sequence during recombination along with increased H4 acetylation, indicating a direct role during strand invasion/D-loop formation after resection. We found that NuA4 cooperates locally with another HAT, the SAGA complex, during DSB repair as their combined action is essential for DNA end resection to occur. This cooperation of NuA4 and SAGA is required for recruitment of ATP-dependent chromatin remodelers, targeted acetylation of repair factors and homologous recombination. Our work reveals a multifaceted and conserved cooperation mechanism between acetyltransferase complexes to allow repair of DNA breaks by homologous recombination. DNA double-strand breaks (DSBs) are among the most dangerous types of DNA lesions as they can produce genomic instability that leads to cancer and genetic diseases. It is therefore crucial to understand the precise molecular mechanisms used by cells to detect and repair this type of damages. Homologous recombination using sister chromatid as template is the most accurate pathway to repair these breaks but has to occur within the context of the DNA compacted structure in chromosomes. Here, we show that two enzymes, NuA4 and SAGA, that acetylate the structural components of chromosomes in the vicinity of the DNA breaks are together essential for recombination-mediated repair to occur. We found that they are recruited at an early step after damage detection and their action allows subsequent remodeling of local structural organisation by other enzymes, providing DNA access to the recombination machinery. These results highlight the cooperation of enzymes for a same goal, providing robustness in the repair process as only the loss of both leads to major defects.
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Affiliation(s)
- Xue Cheng
- St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Division of CHU de Québec-Université Laval Research Center, Quebec City, Canada
| | - Valérie Côté
- St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Division of CHU de Québec-Université Laval Research Center, Quebec City, Canada
| | - Jacques Côté
- St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Division of CHU de Québec-Université Laval Research Center, Quebec City, Canada
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43
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Wong RP, Petriukov K, Ulrich HD. Daughter-strand gaps in DNA replication - substrates of lesion processing and initiators of distress signalling. DNA Repair (Amst) 2021; 105:103163. [PMID: 34186497 DOI: 10.1016/j.dnarep.2021.103163] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2021] [Revised: 06/18/2021] [Accepted: 06/19/2021] [Indexed: 10/21/2022]
Abstract
Dealing with DNA lesions during genome replication is particularly challenging because damaged replication templates interfere with the progression of the replicative DNA polymerases and thereby endanger the stability of the replisome. A variety of mechanisms for the recovery of replication forks exist, but both bacteria and eukaryotic cells also have the option of continuing replication downstream of the lesion, leaving behind a daughter-strand gap in the newly synthesized DNA. In this review, we address the significance of these single-stranded DNA structures as sites of DNA damage sensing and processing at a distance from ongoing genome replication. We describe the factors controlling the emergence of daughter-strand gaps from stalled replication intermediates, the benefits and risks of their expansion and repair via translesion synthesis or recombination-mediated template switching, and the mechanisms by which they activate local as well as global replication stress signals. Our growing understanding of daughter-strand gaps not only identifies them as targets of fundamental genome maintenance mechanisms, but also suggests that proper control over their activities has important practical implications for treatment strategies and resistance mechanisms in cancer therapy.
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Affiliation(s)
- Ronald P Wong
- Institute of Molecular Biology (IMB) gGmbH, Ackermannweg 4, D - 55128 Mainz, Germany
| | - Kirill Petriukov
- Institute of Molecular Biology (IMB) gGmbH, Ackermannweg 4, D - 55128 Mainz, Germany
| | - Helle D Ulrich
- Institute of Molecular Biology (IMB) gGmbH, Ackermannweg 4, D - 55128 Mainz, Germany.
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44
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Rtt105 promotes high-fidelity DNA replication and repair by regulating the single-stranded DNA-binding factor RPA. Proc Natl Acad Sci U S A 2021; 118:2106393118. [PMID: 34140406 DOI: 10.1073/pnas.2106393118] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Single-stranded DNA (ssDNA) covered with the heterotrimeric Replication Protein A (RPA) complex is a central intermediate of DNA replication and repair. How RPA is regulated to ensure the fidelity of DNA replication and repair remains poorly understood. Yeast Rtt105 is an RPA-interacting protein required for RPA nuclear import and efficient ssDNA binding. Here, we describe an important role of Rtt105 in high-fidelity DNA replication and recombination and demonstrate that these functions of Rtt105 primarily depend on its regulation of RPA. The deletion of RTT105 causes elevated spontaneous DNA mutations with large duplications or deletions mediated by microhomologies. Rtt105 is recruited to DNA double-stranded break (DSB) ends where it promotes RPA assembly and homologous recombination repair by gene conversion or break-induced replication. In contrast, Rtt105 attenuates DSB repair by the mutagenic single-strand annealing or alternative end joining pathway. Thus, Rtt105-mediated regulation of RPA promotes high-fidelity replication and recombination while suppressing repair by deleterious pathways. Finally, we show that the human RPA-interacting protein hRIP-α, a putative functional homolog of Rtt105, also stimulates RPA assembly on ssDNA, suggesting the conservation of an Rtt105-mediated mechanism.
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45
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Pham N, Yan Z, Yu Y, Faria Afreen M, Malkova A, Haber JE, Ira G. Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks. EMBO J 2021; 40:e104847. [PMID: 33844333 DOI: 10.15252/embj.2020104847] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 03/04/2021] [Accepted: 03/09/2021] [Indexed: 11/09/2022] Open
Abstract
DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two-ended DNA double-strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single-ended DSBs are repaired by break-induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two-ended DSBs. Here, we demonstrate that BIR is suppressed at two-ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D-loop unwinding helicase Mph1, and (iii) Mre11-Rad50-Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans.
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Affiliation(s)
- Nhung Pham
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Zhenxin Yan
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Yang Yu
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Mosammat Faria Afreen
- Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA
| | - Anna Malkova
- Department of Biology, University of Iowa, Iowa City, IA, USA
| | - James E Haber
- Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA
| | - Grzegorz Ira
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
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46
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A Conserved Histone H3-H4 Interface Regulates DNA Damage Tolerance and Homologous Recombination during the Recovery from Replication Stress. Mol Cell Biol 2021; 41:MCB.00044-20. [PMID: 33526454 DOI: 10.1128/mcb.00044-20] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2020] [Accepted: 01/24/2021] [Indexed: 12/20/2022] Open
Abstract
In eukaryotes, genomic DNA is packaged into nucleosomes, which are the basal components coordinating both the structures and functions of chromatin. In this study, we screened a collection of mutations for histone H3/H4 mutants in Saccharomyces cerevisiae that affect the DNA damage sensitivity of DNA damage tolerance (DDT)-deficient cells. We identified a class of histone H3/H4 mutations that suppress methyl methanesulfonate (MMS) sensitivity of DDT-deficient cells (referred to here as the histone SDD mutations), which likely cluster on a specific H3-H4 interface of the nucleosomes. The histone SDD mutations did not suppress the MMS sensitivity of DDT-deficient cells in the absence of Rad51, indicating that homologous recombination (HR) is responsible for DNA damage resistance. Furthermore, the histone SDD mutants showed reduced levels of PCNA ubiquitination after exposure to MMS or UV irradiation, consistent with decreased MMS-induced mutagenesis relative to that of wild-type cells. We also found that histone SDD mutants lacking the INO80 chromatin remodeler impair HR-dependent recovery from MMS-induced replication arrest, resulting in defective S-phase progression and increased Rad52 foci. Taken together, our data provide novel insights into nucleosome functions, which link INO80-dependent chromatin remodeling to the regulation of DDT and HR during the recovery from replication blockage.
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47
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Peritore M, Reusswig KU, Bantele SCS, Straub T, Pfander B. Strand-specific ChIP-seq at DNA breaks distinguishes ssDNA versus dsDNA binding and refutes single-stranded nucleosomes. Mol Cell 2021; 81:1841-1853.e4. [PMID: 33651987 DOI: 10.1016/j.molcel.2021.02.005] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2020] [Revised: 12/30/2020] [Accepted: 01/29/2021] [Indexed: 02/07/2023]
Abstract
In a first step of DNA double-strand break (DSB) repair by homologous recombination, DNA ends are resected such that single-stranded DNA (ssDNA) overhangs are generated. ssDNA is specifically bound by RPA and other factors, which constitutes a ssDNA-domain on damaged chromatin. The molecular organization of this ssDNA and the adjacent dsDNA domain is crucial during DSB signaling and repair. However, data regarding the presence of nucleosomes, the most basic chromatin components, in the ssDNA domain have been contradictory. Here, we use site-specific induction of DSBs and chromatin immunoprecipitation followed by strand-specific sequencing to analyze in vivo binding of key DSB repair and signaling proteins to either the ssDNA or dsDNA domain. In the case of nucleosomes, we show that recently proposed ssDNA nucleosomes are not a major, persistent species, but that nucleosome eviction and DNA end resection are intrinsically coupled. These results support a model of separated dsDNA-nucleosome and ssDNA-RPA domains during DSB repair.
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Affiliation(s)
- Martina Peritore
- Research Group DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
| | - Karl-Uwe Reusswig
- Research Group DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
| | - Susanne C S Bantele
- Research Group DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
| | - Tobias Straub
- Biomedizinisches Centrum, Core Facility Bioinformatics, Ludwig-Maximilians-Universität München, 82152 Martinsried, Germany
| | - Boris Pfander
- Research Group DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
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48
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RPA-mediated recruitment of Bre1 couples histone H2B ubiquitination to DNA replication and repair. Proc Natl Acad Sci U S A 2021; 118:2017497118. [PMID: 33602814 DOI: 10.1073/pnas.2017497118] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
The ubiquitin E3 ligase Bre1-mediated H2B monoubiquitination (H2Bub) is essential for proper DNA replication and repair in eukaryotes. Deficiency in H2Bub causes genome instability and cancer. How the Bre1-H2Bub pathway is evoked in response to DNA replication or repair remains unknown. Here, we identify that the single-stranded DNA (ssDNA) binding factor RPA acts as a key mediator that couples Bre1-mediated H2Bub to DNA replication and repair in yeast. We found that RPA interacts with Bre1 in vitro and in vivo, and this interaction is stimulated by ssDNA. This association ensures the recruitment of Bre1 to replication forks or DNA breaks but does not affect its E3 ligase activity. Disruption of the interaction abolishes the local enrichment of H2Bub, resulting in impaired DNA replication, response to replication stress, and repair by homologous recombination, accompanied by increased genome instability and DNA damage sensitivity. Notably, we found that RNF20, the human homolog of Bre1, interacts with RPA70 in a conserved mode. Thus, RPA functions as a master regulator for the spatial-temporal control of H2Bub chromatin landscape during DNA replication and recombination, extending the versatile roles of RPA in guarding genome stability.
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49
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Xing P, Dong Y, Zhao J, Zhou Z, Li Z, Wang Y, Li M, Zhang X, Chen X. Mrc1-Dependent Chromatin Compaction Represses DNA Double-Stranded Break Repair by Homologous Recombination Upon Replication Stress. Front Cell Dev Biol 2021; 9:630777. [PMID: 33681209 PMCID: PMC7928320 DOI: 10.3389/fcell.2021.630777] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2020] [Accepted: 01/06/2021] [Indexed: 11/13/2022] Open
Abstract
The coordination of DNA replication and repair is critical for the maintenance of genome stability. It has been shown that the Mrc1-mediated S phase checkpoint inhibits DNA double-stranded break (DSB) repair through homologous recombination (HR). How the replication checkpoint inhibits HR remains only partially understood. Here we show that replication stress induces the suppression of both Sgs1/Dna2- and Exo1-mediated resection pathways in an Mrc1-dependent manner. As a result, the loading of the single-stranded DNA binding factor replication protein A (RPA) and Rad51 and DSB repair by HR were severely impaired under replication stress. Notably, the deletion of MRC1 partially restored the recruitment of resection enzymes, DSB end resection, and the loading of RPA and Rad51. The role of Mrc1 in inhibiting DSB end resection is independent of Csm3, Tof1, or Ctf4. Mechanistically, we reveal that replication stress induces global chromatin compaction in a manner partially dependent on Mrc1, and this chromatin compaction limits the access of chromatin remodeling factors and HR proteins, leading to the suppression of HR. Our study reveals a critical role of the Mrc1-dependent chromatin structure change in coordinating DNA replication and recombination under replication stress.
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Affiliation(s)
- Poyuan Xing
- Hubei Key Laboratory of Cell Homeostasis and the Institute for Advanced Studies, College of Life Sciences, Wuhan University, Wuhan, China
| | - Yang Dong
- Hubei Key Laboratory of Cell Homeostasis and the Institute for Advanced Studies, College of Life Sciences, Wuhan University, Wuhan, China
| | - Jingyu Zhao
- Hubei Key Laboratory of Cell Homeostasis and the Institute for Advanced Studies, College of Life Sciences, Wuhan University, Wuhan, China
| | - Zhou Zhou
- Hubei Key Laboratory of Cell Homeostasis and the Institute for Advanced Studies, College of Life Sciences, Wuhan University, Wuhan, China
| | - Zhao Li
- Hubei Key Laboratory of Cell Homeostasis and the Institute for Advanced Studies, College of Life Sciences, Wuhan University, Wuhan, China
| | - Yu Wang
- Hubei Key Laboratory of Cell Homeostasis and the Institute for Advanced Studies, College of Life Sciences, Wuhan University, Wuhan, China
| | - Mengfei Li
- Hubei Key Laboratory of Cell Homeostasis and the Institute for Advanced Studies, College of Life Sciences, Wuhan University, Wuhan, China
| | - Xinghua Zhang
- Hubei Key Laboratory of Cell Homeostasis and the Institute for Advanced Studies, College of Life Sciences, Wuhan University, Wuhan, China
| | - Xuefeng Chen
- Hubei Key Laboratory of Cell Homeostasis and the Institute for Advanced Studies, College of Life Sciences, Wuhan University, Wuhan, China
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50
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Peng H, Zhang S, Chen X. Monitoring 5'-End Resection at Site-Specific Double-Strand Breaks by Southern Blot Analysis. Methods Mol Biol 2021; 2196:245-255. [PMID: 32889727 DOI: 10.1007/978-1-0716-0868-5_20] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
DNA double-strand break (DSB) is one of the most deleterious types of DNA lesions threatening genome integrity. Cells have evolved several exquisite pathways to repair these breaks. Homologous recombination (HR) is an essential DSB repair mechanism that utilizes an intact homologous sequence as a template to repair DSBs with high fidelity. To initiate the HR repair, the 5'-ends of DSBs have to be nucleolytically cleaved by nucleases to generate 3'-single-strand DNA (ssDNA). Exposed 3'-ssDNA recruits the ssDNA binding protein complex RPA to activate the DNA damage checkpoint. RPA is subsequently replaced by Rad51 recombinase to form Rad51 nucleoprotein filament that catalyzes strand invasion and formation of the D-loop. Processing of 5'-ends (called resection) is a crucial step that determines the choice of repair pathways. Here we introduce an assay for monitoring the dynamics of resection at different locations from a site-specific DSB in yeast.
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Affiliation(s)
- Haoyang Peng
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences and the Institute for Advanced Studies, Wuhan University, Wuhan, China
| | - Simin Zhang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences and the Institute for Advanced Studies, Wuhan University, Wuhan, China
| | - Xuefeng Chen
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences and the Institute for Advanced Studies, Wuhan University, Wuhan, China.
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