Mechanism and significance of chromosome damage repair by homologous recombination

Abstract Homologous recombination (HR) is a major, conserved pathway of chromosome damage repair. It not only fulfills key functions in the removal of deleterious lesions such as DNA double-strand breaks (DSBs) and interstrand cross-links (ICLs), but also in replication fork repair and protection. Several familial and acquired cancer predisposition syndromes stem from defects in HR. In particular, individuals with mutations in HR genes exhibit predisposition to breast, ovarian, pancreatic, and prostate cancers, and they also show signs of accelerated aging. However, aberrant and untimely HR events can lead to the loss of heterozygosity, genomic rearrangements, and cytotoxic nucleoprotein intermediates. Thus, it is critically important that HR be tightly regulated. In addition to DNA repair, HR is also involved in meiotic chromosome segregation and telomere maintenance in cells that lack telomerase. In this review, we focus on the role of HR in DSB repair (DSBR) and summarize the current state of the field.

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XPF-ERCC1 Participates in the Fanconi Anemia Pathway of Cross-Link Repair

Molecular and Cellular Biology ◽

10.1128/mcb.00086-09 ◽

2009 ◽

Vol 29 (24) ◽

pp. 6427-6437 ◽

Cited By ~ 91

Author(s):

Nikhil Bhagwat ◽

Anna L. Olsen ◽

Anderson T. Wang ◽

Katsuhiro Hanada ◽

Patricia Stuckert ◽

...

Keyword(s):

Homologous Recombination ◽

Fanconi Anemia ◽

Dna Double Strand Breaks ◽

Fanconi Anemia Pathway ◽

Strand Breaks ◽

Interstrand Cross Links ◽

Mammalian Genomes ◽

Cross Links ◽

Dna Strand ◽

Transcription And Replication

ABSTRACT Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81 −/−, Ercc1 −/−, and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.

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DNA Interstrand Cross-Link Repair in the Saccharomyces cerevisiae Cell Cycle: Overlapping Roles for PSO2 (SNM1) with MutS Factors and EXO1 during S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.25.6.2297-2309.2005 ◽

2005 ◽

Vol 25 (6) ◽

pp. 2297-2309 ◽

Cited By ~ 51

Author(s):

Louise J. Barber ◽

Thomas A. Ward ◽

John A. Hartley ◽

Peter J. McHugh

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Homologous Recombination ◽

S Phase ◽

Cycle Phase ◽

Dna Double Strand Breaks ◽

Recombination Rates ◽

Strand Breaks ◽

Interstrand Cross Links ◽

Cross Links

ABSTRACT Pso2/Snm1 is a member of the β-CASP metallo-β-lactamase family of proteins that include the V(D)J recombination factor Artemis. Saccharomyces cerevisiae pso2 mutants are specifically sensitive to agents that induce DNA interstrand cross-links (ICLs). Here we establish a novel overlapping function for PSO2 with MutS mismatch repair factors and the 5′-3′ exonuclease Exo1 in the repair of DNA ICLs, which is confined to S phase. Our data demonstrate a requirement for NER and Pso2, or Exo1 and MutS factors, in the processing of ICLs, and this is required prior to the repair of ICL-induced DNA double-strand breaks (DSBs) that form during replication. Using a chromosomally integrated inverted-repeat substrate, we also show that loss of both pso2 and exo1/msh2 reduces spontaneous homologous recombination rates. Therefore, PSO2, EXO1, and MSH2 also appear to have overlapping roles in the processing of some forms of endogenous DNA damage that occur at an irreversibly collapsed replication fork. Significantly, our analysis of ICL repair in cells synchronized for each cell cycle phase has revealed that homologous recombination does not play a major role in the direct repair of ICLs, even in G2, when a suitable template is readily available. Rather, we propose that recombination is primarily involved in the repair of DSBs that arise from the collapse of replication forks at ICLs. These findings have led to considerable clarification of the complex genetic relationship between various ICL repair pathways.

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The Structure-Specific Endonuclease Ercc1-Xpf Is Required To Resolve DNA Interstrand Cross-Link-Induced Double-Strand Breaks

Molecular and Cellular Biology ◽

10.1128/mcb.24.13.5776-5787.2004 ◽

2004 ◽

Vol 24 (13) ◽

pp. 5776-5787 ◽

Cited By ~ 348

Author(s):

Laura J. Niedernhofer ◽

Hanny Odijk ◽

Magda Budzowska ◽

Ellen van Drunen ◽

Alex Maas ◽

...

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Cell Cycle Progression ◽

Strand Break ◽

Sister Chromatid Exchanges ◽

Wild Type ◽

Cross Link ◽

Interstrand Cross Links ◽

Normal Metabolism ◽

Cross Links

ABSTRACT Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (γ-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced γ-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, γ-H2AX foci were also induced in Ercc1 −/− cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1 −/− cells, MMC-induced γ-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1 −/− and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.

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Regulatory control of Sgs1 and Dna2 during eukaryotic DNA end resection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1819276116 ◽

2019 ◽

Vol 116 (13) ◽

pp. 6091-6100 ◽

Cited By ~ 12

Author(s):

Chaoyou Xue ◽

Weibin Wang ◽

J. Brooks Crickard ◽

Corentin J. Moevus ◽

Youngho Kwon ◽

...

Keyword(s):

Homologous Recombination ◽

Single Molecule ◽

Protein A ◽

Regulatory Control ◽

Dna Double Strand Breaks ◽

Nucleosome Remodeling ◽

Damage Repair ◽

Eukaryotic Dna ◽

Dna End Resection ◽

Dna Ends

In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3′ single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1–dependent or –independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1–Dna2–Top3-Rmi1–RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.

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Involvement of Nucleotide Excision Repair in a Recombination-Independent and Error-Prone Pathway of DNA Interstrand Cross-Link Repair

Molecular and Cellular Biology ◽

10.1128/mcb.21.3.713-720.2001 ◽

2001 ◽

Vol 21 (3) ◽

pp. 713-720 ◽

Cited By ~ 94

Author(s):

Xin Wang ◽

Carolyn A. Peterson ◽

Huyong Zheng ◽

Rodney S. Nairn ◽

Randy J. Legerski ◽

...

Keyword(s):

Homologous Recombination ◽

Nucleotide Excision Repair ◽

Mammalian Cells ◽

Cultured Cells ◽

Excision Repair ◽

Repair Pathway ◽

Cross Link ◽

Nucleotide Excision ◽

Interstrand Cross Links ◽

Cross Links

ABSTRACT DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.

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Depurination of colibactin-derived interstrand cross-links

10.1101/869313 ◽

2019 ◽

Author(s):

Mengzhao Xue ◽

Kevin M. Wernke ◽

Seth B. Herzon

Keyword(s):

Cancer Progression ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Dna Dsbs ◽

Interstrand Cross Links ◽

Cross Links ◽

Non Homologous End Joining ◽

Colorectal Cancer Progression

AbstractColibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer progression. Evidence suggests colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we show that colibactin-derived ICLs are unstable toward depurination and elimination of the 3′ phosphate. This pathway leads progressively to the formation of nicks SSBs and cleavage DSBs and is consistent with the earlier determination that non-homologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein further our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.

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Lamin A/C: Function in Normal and Tumor Cells

Cancers ◽

10.3390/cancers12123688 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3688

Author(s):

Niina Dubik ◽

Sabine Mai

Keyword(s):

Nuclear Protein ◽

Striated Muscle ◽

Accelerated Aging ◽

Lamin A ◽

Muscle Disorders ◽

Damage Repair ◽

Telomere Protection ◽

Aggressive Cancer ◽

Prostate Cancers ◽

Mechanical Insult

This review is focused on lamin A/C, a nuclear protein with multiple functions in normal and diseased cells. Its functions, as known to date, are summarized. This summary includes its role in maintaining a cell’s structural stability, cell motility, mechanosensing, chromosome organization, gene regulation, cell differentiation, DNA damage repair, and telomere protection. As lamin A/C has a variety of critical roles within the cell, mutations of the lamin A/C gene and incorrect processing of the protein results in a wide variety of diseases, ranging from striated muscle disorders to accelerated aging diseases. These diseases, collectively termed laminopathies, are also touched upon. Finally, we review the existing evidence of lamin A/C’s deregulation in cancer. Lamin A/C deregulation leads to various traits, including genomic instability and increased tolerance to mechanical insult, which can lead to more aggressive cancer and poorer prognosis. As lamin A/C’s expression in specific cancers varies widely, currently known lamin A/C expression in various cancers is reviewed. Additionally, Lamin A/C’s potential as a biomarker in various cancers and as an aid in more accurately diagnosing intermediate Gleason score prostate cancers is also discussed.

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The BRCA Tumor Suppressor Network in Chromosome Damage Repair by Homologous Recombination

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-013118-111058 ◽

2019 ◽

Vol 88 (1) ◽

pp. 221-245 ◽

Cited By ~ 25

Author(s):

Weixing Zhao ◽

Claudia Wiese ◽

Youngho Kwon ◽

Robert Hromas ◽

Patrick Sung

Keyword(s):

Tumor Suppressor ◽

Homologous Recombination ◽

Cancer Therapeutics ◽

Recent Effort ◽

Chromosome Damage ◽

Replication Forks ◽

Genome Maintenance ◽

Brca1 And Brca2 ◽

Damage Repair ◽

Structural Insights

Mutations in the BRCA1 and BRCA2 genes predispose afflicted individuals to breast, ovarian, and other cancers. The BRCA-encoded products form complexes with other tumor suppressor proteins and with the recombinase enzyme RAD51 to mediate chromosome damage repair by homologous recombination and also to protect stressed DNA replication forks against spurious nucleolytic attrition. Understanding how the BRCA tumor suppressor network executes its biological functions would provide the foundation for developing targeted cancer therapeutics, but progress in this area has been greatly hampered by the challenge of obtaining purified BRCA complexes for mechanistic studies. In this article, we review how recent effort begins to overcome this technical challenge, leading to functional and structural insights into the biochemical attributes of these complexes and the multifaceted roles that they fulfill in genome maintenance. We also highlight the major mechanistic questions that remain.

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Differential Roles for DNA Polymerases Eta, Zeta, and REV1 in Lesion Bypass of Intrastrand versus Interstrand DNA Cross-Links

Molecular and Cellular Biology ◽

10.1128/mcb.00993-09 ◽

2009 ◽

Vol 30 (5) ◽

pp. 1217-1230 ◽

Cited By ~ 89

Author(s):

J. Kevin Hicks ◽

Colleen L. Chute ◽

Michelle T. Paulsen ◽

Ryan L. Ragland ◽

Niall G. Howlett ◽

...

Keyword(s):

Dna Polymerases ◽

Dna Lesions ◽

Dna Double Strand Breaks ◽

Lesion Bypass ◽

Translesion Dna Synthesis ◽

Strand Breaks ◽

Cycle Arrest ◽

Interstrand Cross Links ◽

Cross Links ◽

Translesion Dna

ABSTRACT Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Polη), REV1, and zeta (Polζ) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Polζ are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Polζ facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Polη, whereas RAD18 plus Polη, REV1, and Polζ are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links.

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