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. 2012 Jan;40(2):682-91.
doi: 10.1093/nar/gkr769. Epub 2011 Sep 16.

REV1 and Polymerase ζ Facilitate Homologous Recombination Repair

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Free PMC article

REV1 and Polymerase ζ Facilitate Homologous Recombination Repair

Shilpy Sharma et al. Nucleic Acids Res. .
Free PMC article

Abstract

REV1 and DNA Polymerase ζ (REV3 and REV7) play important roles in translesion DNA synthesis (TLS) in which DNA replication bypasses blocking lesions. REV1 and Polζ have also been implicated in promoting repair of DNA double-stranded breaks (DSBs). However, the mechanism by which these two TLS polymerases increase tolerance to DSBs is poorly understood. Here we demonstrate that full-length human REV1, REV3 and REV7 interact in vivo (as determined by co-immunoprecipitation studies) and together, promote homologous recombination repair. Cells lacking REV3 were hypersensitive to agents that cause DSBs including the PARP inhibitor, olaparib. REV1, REV3 or REV7-depleted cells displayed increased chromosomal aberrations, residual DSBs and sites of HR repair following exposure to ionizing radiation. Notably, cells depleted of DNA polymerase η (Polη) or the E3 ubiquitin ligase RAD18 were proficient in DSB repair following exposure to IR indicating that Polη-dependent lesion bypass or RAD18-dependent monoubiquitination of PCNA are not necessary to promote REV1 and Polζ-dependent DNA repair. Thus, the REV1/Polζ complex maintains genomic stability by directly participating in DSB repair in addition to the canonical TLS pathway.

Figures

Figure 1.
Figure 1.
REV1, REV3, and REV7 form a ternary complex. 293T cells were transfected with a plasmid encoding REV3-Flag and a control plasmid or combinations of REV3-Flag with GFP-REV1 or GFP-REV7 encoding plasmids. Cell lysates were treated with benzonase, pre-cleared, and then subjected to immunoprecipitation and immunoblotting with the indicated antibodies. (A) REV3-Flag immunoprecipitates contain GFP-REV1 and endogenous REV7 (left) or GFP-REV7 and endongenous REV1 (right). (B) GFP-REV1 immunoprecipitates contain REV3-Flag (left). GFP-REV7 immunoprecipites contain REV3-Flag and endogenous REV1 (right). (C) REV3-Flag immunoprecipitates contain both endogenous REV1 and REV7. Specificity of REV1 and REV7 antibodies are demonstrated by the depletion of each protein with gene-specific siRNA co-transfected with REV3-Flag expression vector.
Figure 2.
Figure 2.
REV1 or Polζ (REV3 and REV7) depletion leads to hypersensitivity to ionizing radiation and a defect in DSB repair. HeLa cells were transfected with control siRNA (Control) or the indicated siRNAs targeting a specific DNA polymerase gene and assessed for their response to IR. (A) Depletion of REV1, REV3, or REV7 is associated with a prolonged IR-induced G2 cell cycle checkpoint. Cells were exposed to 0 or 4 Gy IR and subjected to cell cycle analysis 24 h later by flow cytometry. (B) Sensitivity to IR was measured using a standard clonogenic survival assay. Data are presented as the mean ± S.E.M from three independent experiments. (C) siRNA transfected HeLa cells were examined for the presence of chromosomal aberrations (gaps and breaks) 24 h after 2 or 4 Gy IR. Data from a representative experiment is expressed as the average number of gaps and breaks per metaphase ± SEM (n = 50). (D) HeLa cells transfected with the indicated siRNAs were exposed to 2 Gy IR and then fixed 24 h later. Cells were immunostained with S1981P-ATM and 53BP1 to mark sites of DSBs. The percentage of cells displaying 10 or more colocalized foci is shown as the mean ± SEM from three independent experiments. Representative images are shown in (E) and additional quantification data are shown in Supplementary Figure S5. HeLa cells transfected with RAD51-specific siRNA (HR-repair defective) are shown for comparative purposes in (C–E).
Figure 3.
Figure 3.
REV1 and Polζ promote HR repair. HeLa cells containing the DR-GFP reporter construct (A) integrated into the genome were transfected twice with the indicated siRNAs. The next day, cells were infected with adenovirus AdNGUS24i expressing the I-SceI enzyme. (B) Two days later, cells were assessed for GFP expression by flow cytometry. Control adenovirus did not promote detectable expression of GFP protein (data not shown). (C and D) U2OS or SV40-imortalized human fibroblasts containing the DR-GFP reporter were transfected twice with the inidicated siRNAs and analyzed as in (B). (E and F) REV1, REV3 or REV7-depleted HeLa cells display elevated numbers of RAD51 or RPA/p34 foci 24 h after exposure to 2 Gy IR. The percentage of cells exhibiting 10 or more RAD51 foci (E) or RPA/p34 (F) is shown for each siRNA. Representative images of cells scoring positive (REV3-2 transfected cells treated with 2 Gy IR) are shown. (B through F) Data are the mean ± SEM of at least three independent experiments.
Figure 4.
Figure 4.
REV1 and Polζ promote radioresistance independently of RAD18. (A and B) HeLa cells transfected with siRNAs targeting RAD18 or RAD51 were exposed to 4 J/m2 UV-C light or 4 Gy IR as indicated. Twenty-four hours later, cells were subjected to cell cycle analysis by flow cytometry to measure DNA content per cell. (C) HeLa cells depleted of RAD18 stain intensely for γ-H2AX 24 h after exposure to UV-C irradiation indicative of strong DNA damage response due to replication fork stalling and efficient protein knockdown. (D) RAD18-depleted HeLa cells are proficient in resolving foci containing S1981P-ATM and 53BP1 within 24 h. Cells transfected with RAD51 siRNA were analyzed as a positive control for HR repair deficiency. The mean percentage of cells exhibiting 10 or more co-localized foci is shown in (E). Data are the mean ± SEM of three independent experiments.
Figure 5.
Figure 5.
Deletion of the REV3L gene leads to hypersensitivity to agents that induce DSBs. Parental BL2 cells (WT) and BL2 cells deleted of the indicated genes were treated with various doses of DNA damaging agent and harvested 48 (IR, Neocarzinostatin, H2O2) or 72 h (etoposide, camptothecin, olaparib) later. Viability was determined by measuring the percentage of cells excluding propidium iodide by flow cytometry. Data represent the mean ± SEM of at least three independent experiments.

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References

    1. Sung P, Klein H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat. Rev. Mol. Cell. Biol. 2006;7:739–750. - PubMed
    1. Moynahan ME, Jasin M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell. Biol. 2010;11:196–207. - PMC - PubMed
    1. Hicks WM, Kim M, Haber JE. Increased mutagenesis and unique mutation signature associated with mitotic gene conversion. Science. 2010;329:82–85. - PMC - PubMed
    1. Smith CE, Lam AF, Symington LS. Aberrant double-strand break repair resulting in half crossovers in mutants defective for Rad51 or the DNA polymerase δ Complex. Mol. Cell. Biol. 2009;29:1432–1441. - PMC - PubMed
    1. Lydeard JR, Jain S, Yamaguchi M, Haber JE. Break-induced replication and telomerase-independent telomere maintenance require Pol32. Nature. 2007;448:820–823. - PubMed

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