Fidelity of Mitotic Double-Strand-Break Repair in Saccharomyces cerevisiae: A Role for SAE2/COM1

Abstract Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is ∼100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an ∼10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Δ strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.

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The Roles of REV3 and RAD57 in Double-Strand-Break-Repair-Induced Mutagenesis of Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/162.3.1063 ◽

2002 ◽

Vol 162 (3) ◽

pp. 1063-1077 ◽

Cited By ~ 1

Author(s):

Alison J Rattray ◽

Brenda K Shafer ◽

Carolyn B McGill ◽

Jeffrey N Strathern

Keyword(s):

Dna Synthesis ◽

Point Mutations ◽

Fold Increase ◽

Strand Break ◽

Base Substitution ◽

Recombinational Repair ◽

Induced Mutations ◽

In The Wild ◽

Break Repair ◽

Substitution Mutations

Abstract The DNA synthesis associated with recombinational repair of chromosomal double-strand breaks (DSBs) has a lower fidelity than normal replicative DNA synthesis. Here, we use an inverted-repeat substrate to monitor the fidelity of repair of a site-specific DSB. DSB induction made by the HO endonuclease stimulates recombination >5000-fold and is associated with a >1000-fold increase in mutagenesis of an adjacent gene. We demonstrate that most break-repair-induced mutations (BRIMs) are point mutations and have a higher proportion of frameshifts than do spontaneous mutations of the same substrate. Although the REV3 translesion DNA polymerase is not required for recombination, it introduces ∼75% of the BRIMs and ∼90% of the base substitution mutations. Recombinational repair of the DSB is strongly dependent upon genes of the RAD52 epistasis group; however, the residual recombinants present in rad57 mutants are associated with a 5- to 20-fold increase in BRIMs. The spectrum of mutations in rad57 mutants is similar to that seen in the wild-type strain and is similarly affected by REV3. We also find that REV3 is required for the repair of MMS-induced lesions when recombinational repair is compromised. Our data suggest that Rad55p/Rad57p help limit the generation of substrates that require pol ζ during recombination.

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Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is Dependent on Its Association with Ku80

Molecular and Cellular Biology ◽

10.1128/mcb.01075-08 ◽

2008 ◽

Vol 28 (24) ◽

pp. 7380-7393 ◽

Cited By ~ 45

Author(s):

Leizhen Wei ◽

Li Lan ◽

Zehui Hong ◽

Akira Yasui ◽

Chikashi Ishioka ◽

...

Keyword(s):

Susceptibility Gene ◽

Repair Process ◽

Strand Break ◽

Missense Mutations ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Nhej Pathway ◽

C Terminus ◽

N Terminus

ABSTRACT BRCA1 is the first susceptibility gene to be linked to breast and ovarian cancers. Although mounting evidence has indicated that BRCA1 participates in DNA double-strand break (DSB) repair pathways, its precise mechanism is still unclear. Here, we analyzed the in situ response of BRCA1 at DSBs produced by laser microirradiation. The amino (N)- and carboxyl (C)-terminal fragments of BRCA1 accumulated independently at DSBs with distinct kinetics. The N-terminal BRCA1 fragment accumulated immediately after laser irradiation at DSBs and dissociated rapidly. In contrast, the C-terminal fragment of BRCA1 accumulated more slowly at DSBs but remained at the sites. Interestingly, rapid accumulation of the BRCA1 N terminus, but not the C terminus, at DSBs depended on Ku80, which functions in the nonhomologous end-joining (NHEJ) pathway, independently of BARD1, which binds to the N terminus of BRCA1. Two small regions in the N terminus of BRCA1 independently accumulated at DSBs and interacted with Ku80. Missense mutations found within the N terminus of BRCA1 in cancers significantly changed the kinetics of its accumulation at DSBs. A P142H mutant failed to associate with Ku80 and restore resistance to irradiation in BRCA1-deficient cells. These might provide a molecular basis of the involvement of BRCA1 in the NHEJ pathway of the DSB repair process.

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The Chromosome Bias of Misincorporations During Double-Strand Break Repair Is Not Altered in Mismatch Repair–Defective Strains of Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/148.4.1525 ◽

1998 ◽

Vol 148 (4) ◽

pp. 1525-1533 ◽

Cited By ~ 3

Author(s):

Carolyn B McGill ◽

Susan L Holbeck ◽

Jeffrey N Strathern

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Synthesis ◽

Mismatch Repair ◽

Strand Break ◽

Recombinational Repair ◽

Dsb Repair ◽

Site Specific ◽

Initial Sequence ◽

Dna Break ◽

A Site

AbstractRecombinational repair of a site-specific, double-strand DNA break (DSB) results in increased reversion frequency for nearby mutations. Although some models for DSB repair predict that newly synthesized DNA will be inherited equally by both the originally broken chromosome and the chromosome that served as a template, the DNA synthesis errors are almost exclusively found on the chromosome that had the original DSB (introduced by the HO endonuclease). To determine whether mismatch repair acts on the template chromosome in a directed fashion to restore mismatches to the initial sequence, these experiments were repeated in mismatch repair-defective (pms1, mlh1, and msh2) backgrounds. The results suggest that mismatch repair is not responsible for the observed bias.

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Actin-Related Protein 6 (Arp6) Influences Double-Strand Break Repair in Yeast

Applied Microbiology ◽

10.3390/applmicrobiol1020017 ◽

2021 ◽

Vol 1 (2) ◽

pp. 225-238

Author(s):

Mohsen Hooshyar ◽

Daniel Burnside ◽

Maryam Hajikarimlou ◽

Katayoun Omidi ◽

Alexander Jesso ◽

...

Keyword(s):

Dna Damage ◽

Chromatin Remodeling ◽

Genetic Interaction ◽

Interaction Analysis ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Dna Damaging Agents ◽

Repair Efficiency ◽

Chromosomal Breaks

DNA double-strand breaks (DSBs) are the most deleterious form of DNA damage and are repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR). Repair initiation, regulation and communication with signaling pathways require several histone-modifying and chromatin-remodeling complexes. In budding yeast, this involves three primary complexes: INO80-C, which is primarily associated with HR, SWR1-C, which promotes NHEJ, and RSC-C, which is involved in both pathways as well as the general DNA damage response. Here we identify ARP6 as a factor involved in DSB repair through an RSC-C-related pathway. The loss of ARP6 significantly reduces the NHEJ repair efficiency of linearized plasmids with cohesive ends, impairs the repair of chromosomal breaks, and sensitizes cells to DNA-damaging agents. Genetic interaction analysis indicates that ARP6, MRE11 and RSC-C function within the same pathway, and the overexpression of ARP6 rescues rsc2∆ and mre11∆ sensitivity to DNA-damaging agents. Double mutants of ARP6, and members of the INO80 and SWR1 complexes, cause a significant reduction in repair efficiency, suggesting that ARP6 functions independently of SWR1-C and INO80-C. These findings support a novel role for ARP6 in DSB repair that is independent of the SWR1 chromatin remodeling complex, through an apparent RSC-C and MRE11-associated DNA repair pathway.

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Regulatory and Functional Involvement of Long Non-Coding RNAs in DNA Double-Strand Break Repair Mechanisms

Cells ◽

10.3390/cells10061506 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1506

Author(s):

Angelos Papaspyropoulos ◽

Nefeli Lagopati ◽

Ioanna Mourkioti ◽

Andriani Angelopoulou ◽

Spyridon Kyriazis ◽

...

Keyword(s):

Therapeutic Potential ◽

Strand Break ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Repair Mechanisms ◽

Non Coding Rnas ◽

Repair Pathways ◽

Non Homologous End Joining ◽

Living Organisms

Protection of genome integrity is vital for all living organisms, particularly when DNA double-strand breaks (DSBs) occur. Eukaryotes have developed two main pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR), to repair DSBs. While most of the current research is focused on the role of key protein players in the functional regulation of DSB repair pathways, accumulating evidence has uncovered a novel class of regulating factors termed non-coding RNAs. Non-coding RNAs have been found to hold a pivotal role in the activation of DSB repair mechanisms, thereby safeguarding genomic stability. In particular, long non-coding RNAs (lncRNAs) have begun to emerge as new players with vast therapeutic potential. This review summarizes important advances in the field of lncRNAs, including characterization of recently identified lncRNAs, and their implication in DSB repair pathways in the context of tumorigenesis.

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RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair

10.1101/2021.04.23.440542 ◽

2021 ◽

Author(s):

Takaaki Yasuhara ◽

Reona Kato ◽

Motohiro Yamauchi ◽

Yuki Uchihara ◽

Lee Zou ◽

...

Keyword(s):

Active Regions ◽

Strand Break ◽

Double Strand Breaks ◽

Critical Component ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Chromosome Translocations ◽

Dna Double Strand Break

AbstractR-loops, consisting of ssDNA and DNA-RNA hybrids, are potentially vulnerable unless they are appropriately processed. Recent evidence suggests that R-loops can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. Yet, how the vulnerability of R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops and chromosome translocations and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end-joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing.

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The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

10.1101/2020.07.19.210773 ◽

2020 ◽

Author(s):

Kenta Shinoda ◽

Dali Zong ◽

Elsa Callen ◽

Wei Wu ◽

Lavinia C. Dumitrache ◽

...

Keyword(s):

Dna Damage ◽

Genome Instability ◽

Parp Inhibitor ◽

Progression Free Survival ◽

Strand Break ◽

Transcriptional Network ◽

Cross Resistance ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Protein Levels

AbstractThe Shieldin complex, consisting of SHLD1, SHLD2, SHLD3 and REV7, shields DNA double strand breaks (DSBs) from nucleolytic resection. The end-protecting activity of Shieldin promotes productive non-homologous end joining (NHEJ) in G1 but can threaten genome integrity during S-phase by blocking homologous recombination (HR). Curiously, the penultimate Shieldin component, SHLD1 is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1 and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1. Functionally, this transcriptional network ensures that SHLD1 protein levels are kept in check to enable a proper balance between end protection and end resection during physiological DSB repair. In the context of BRCA1 deficiency, loss of THAP1 dependent SHLD1 expression confers cross resistance to PARP inhibitor and cisplatin, and shorter progression free survival in ovarian cancer patients. In contrast, loss of THAP1 in BRCA2 deficient cells increases genome instability and correlates with improved responses to chemotherapy. Pathogenic THAP1 mutations are causatively linked to the adult-onset torsion dystonia type 6 (DYT6) movement disorder, but the critical disease targets are unknown. We further demonstrate that murine models of Thap1-associated dystonia show reduced Shld1 expression concomitant with elevated levels of unresolved DNA damage in the brain. In summary, our study provides the first example of a transcriptional network that directly controls DSB repair choice and reveals a previously unsuspected link between DNA damage and dystonia.

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Global Epigenetic Analysis Reveals H3K27 Methylation as a Mediator of Double Strand Break Repair

10.1101/2021.09.20.461136 ◽

2021 ◽

Author(s):

Julian Lutze ◽

Donald Wolfgeher ◽

Stephen J. Kron

Keyword(s):

Dna Damage ◽

Cell Fate ◽

Strand Break ◽

Extensive Literature ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Checkpoint Signaling ◽

H3k27 Methylation ◽

Proteomic Data ◽

Global Impacts

AbstractThe majority of cancer patients is treated with ionizing radiation (IR), a relatively safe and effective treatment considered to target tumors by inducing DNA double strand breaks (DSBs). Despite clinical interest in increasing the efficacy of IR by preventing successful DSB repair, few effective radio-adjuvant therapies exist. Extensive literature suggests that chromatin modifiers play a role in the DSB repair and thus may represent a novel class of radiosensitizers. Indeed, chromatin has both local and global impacts on DSB formation, recognition of breaks, checkpoint signaling, recruitment of repair factors, and timely DSB resolution, suggesting that epigenetic deregulation in cancer may impact the efficacy of radiotherapy. Here, using tandem mass spectrometry proteomics to analyze global patterns of histone modification in MCF7 breast cancer cells following IR exposure, we find significant and long-lasting changes to the epigenome. Our results confirm that H3K27 trimethylation (H3K27me3), best known for mediating gene repression and regulating cell fate, increases after IR. H3K27me3 changes rapidly, accumulating at sites of DNA damage. Inhibitors of the Polycomb related complex subunit and H3K27 methyltransferase EZH2 confirm that H3K27me3 is necessary for DNA damage recognition and cell survival after IR. These studies provide an argument for evaluating EZH2 as a radiosensitization target and H3K27me3 as a marker for radiation response in cancer. Proteomic data are available via ProteomeXchange with identifier PXD019388.

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DNA double-strand break repair: a theoretical framework and its application

Journal of The Royal Society Interface ◽

10.1098/rsif.2015.0679 ◽

2016 ◽

Vol 13 (114) ◽

pp. 20150679 ◽

Cited By ~ 9

Author(s):

Philip J. Murray ◽

Bart Cornelissen ◽

Katherine A. Vallis ◽

S. Jon Chapman

Keyword(s):

Dna Damage ◽

Auger Electron ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Histone H2ax ◽

Dna Double Strand Break ◽

A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

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Hedgehog signaling enables repair of ribosomal DNA double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa733 ◽

2020 ◽

Vol 48 (18) ◽

pp. 10342-10352

Author(s):

Tshering D Lama-Sherpa ◽

Victor T G Lin ◽

Brandon J Metge ◽

Shannon E Weeks ◽

Dongquan Chen ◽

...

Keyword(s):

Cancer Cells ◽

Ribosomal Dna ◽

Hedgehog Signaling ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Rdna Sequences ◽

Polymerase I

Abstract Ribosomal DNA (rDNA) consists of highly repeated sequences that are prone to incurring damage. Delays or failure of rDNA double-strand break (DSB) repair are deleterious, and can lead to rDNA transcriptional arrest, chromosomal translocations, genomic losses, and cell death. Here, we show that the zinc-finger transcription factor GLI1, a terminal effector of the Hedgehog (Hh) pathway, is required for the repair of rDNA DSBs. We found that GLI1 is activated in triple-negative breast cancer cells in response to ionizing radiation (IR) and localizes to rDNA sequences in response to both global DSBs generated by IR and site-specific DSBs in rDNA. Inhibiting GLI1 interferes with rDNA DSB repair and impacts RNA polymerase I activity and cell viability. Our findings tie Hh signaling to rDNA repair and this heretofore unknown function may be critically important in proliferating cancer cells.

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