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, 8 (1), 859

Replication Fork Reversal Triggers Fork Degradation in BRCA2-defective Cells

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Replication Fork Reversal Triggers Fork Degradation in BRCA2-defective Cells

Sofija Mijic et al. Nat Commun.

Abstract

Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation. Defective fork stability contributes to chemotherapeutic sensitivity of BRCA2-defective tumors by yet-elusive mechanisms. Using DNA fiber spreading and direct visualization of replication intermediates, we report that reversed replication forks are entry points for fork degradation in BRCA2-defective cells. Besides MRE11 and PTIP, we show that RAD52 promotes stalled fork degradation and chromosomal breakage in BRCA2-defective cells. Inactivation of these factors restores reversed fork frequency and chromosome integrity in BRCA2-defective cells. Conversely, impairing fork reversal prevents fork degradation, but increases chromosomal breakage, uncoupling fork protection, and chromosome stability. We propose that BRCA2 is dispensable for RAD51-mediated fork reversal, but assembles stable RAD51 nucleofilaments on regressed arms, to protect them from degradation. Our data uncover the physiopathological relevance of fork reversal and illuminate a complex interplay of homologous recombination factors in fork remodeling and stability.BRCA2 is involved in both homologous recombination (HR) and the protection of stalled replication forks from degradation. Here the authors reveal how HR factors cooperate in fork remodeling, showing that BRCA2 supports RAD51 loading on the regressed arms of reversed replication forks to protect them from degradation.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Stalled replication forks can reverse in the absence of BRCA2, but are targeted by nucleolytic degradation. a, c Electron micrographs of representative replication forks from RPE-1 cells: parental (P) and daughter (D) duplexes. a The black arrow indicates the regressed arm (R); the four-way junction at the reversed fork is magnified in the inset. c The white arrow points to a ssDNA region at the fork. Scale bar, 200 nm ( = 460 bp), 40 nm ( = 92 bp) in the inset. b Left panel: frequency of reversed replication forks isolated from mock-depleted (siLuc) and BRCA2-depleted (siBRCA2) RPE-1 cells upon optional 5 h treatment with 4 mM HU; where indicated, 50 μM mirin was added 1 h before HU treatment (6 h total treatment). The number of replication intermediates analyzed is indicated in parentheses. The graph depicts mean and SD from three independent EM experiments, blinded to the investigator. The results of the individual biological replicates are in Supplementary Table 1. Right panel: western blot analysis of BRCA2 levels in siLuc and siBRCA2 RPE-1 cells, 48 h after transfection. TFIIH, loading control. d Graphical distribution of ssDNA length at the junction (white arrow in Fig. 1c) in siLuc and siBRCA2 RPE-1 cells optionally treated with 4 mM of HU for 5 h and 50 μM of mirin for 6 h. Only molecules with detectable ssDNA stretches are included in the analysis. The lines show the median length of ssDNA regions at the fork in the specific set of analyzed molecules. Statistical analysis: Mann–Whitney test; ns, not significant; *P < 0.1; **P < 0.01; ***P < 0.001; ****P < 0.0001. The number of analyzed molecules is in brackets
Fig. 2
Fig. 2
BRCA2 maintains reversed fork stability in different cell lines and upon different genotoxic treatments. a Top: schematic representation of BRCA2 protein. Green boxes: RAD51-binding BRC repeats; black box: DBD, DNA-binding domain; C-ter, yellow bar: RAD51-biding C-terminal region. Blue arrows indicate truncations in V-C8 cells; the S3291A mutation is marked in red. Bottom: frequency of reversed replication forks isolated from VC-8 cells and V-C8 cells stably expressing full-length BRCA2 or BRCA2 containing the S3291A mutation, treated as in Fig. 1 (4 mM HU for 5 h; 50 μM mirin for 6 h). The number of analyzed molecules is indicated in brackets. Results of two independent EM experiments are in Supplementary Table 4. Right: western blot analysis of BRCA2 levels in V-C8 and complemented cells. TFIIH, loading control. b EM-based analysis of reversed replication forks isolated from siLuc and siBRCA2 (48 h) RPE-1 cells treated with 25 nM CPT for 1 h; where indicated, 50 μM mirin was added 1 h before CPT treatment (2 h total treatment). In brackets, the total number of analyzed molecules. Results of two independent EM experiments are in Supplementary Table 5
Fig. 3
Fig. 3
Impairing replication fork reversal prevents fork degradation in BRCA2-defective cells. a RPE-1 cells were transfected with siRNA before CldU (red) and IdU (green) labeling (siBRCA2, 48 h; siRAD51, 24 h), followed by treatment with 4 mM HU for 5 h. Left panel: levels of indicated proteins, assessed by western blot. TFIIH, loading control. Middle panel: a representative set of DNA fibers from each condition is shown. Right panel: IdU/CIdU tract length ratio is plotted. Horizontal lines and the numbers indicate the median value. Whiskers indicate the 10–90 percentiles. At least one hundred replication forks were analyzed for each condition. Statistical analysis: Mann–Whitney test; ns, not significant; ****P < 0.0001. See also Supplementary Fig. 4a. b Frequency of reversed replication forks isolated from siLuc, siBRCA2 (48 h) and siRAD51 (24 h) RPE-1 cells treated as in Fig. 1 (4 mM HU for 5 h; 50 μM mirin for 6 h). In brackets, the total number of analyzed molecules. Results of two independent EM experiments are in Supplementary Table 6. c The indicated U2OS-based cell lines were transfected with siRNA before CldU (red) and IdU (green) labeling (siBRCA2, 48 h; siRAD51, 24 h), followed by treatment with 4 mM HU for 5 h and DNA fiber spreading. Left panel: levels of indicated proteins, assessed by western blot. TFIIH, loading control. Right panel: IdU/CIdU tract length ratio is plotted. Track length analysis and statistics as in Fig. 3a. d Stable derivatives of ZRANB3-KO U2OS cells, expressing wild-type (WT), helicase-dead (HD), or nuclease-dead (ND) ZRANB3 at endogenous levels were transfected with siRNA for BRCA2 48 h before CldU (red) and IdU (green) labeling, followed by treatment with 4 mM HU for 5 h. Track length analysis and statistics as in Fig. 3a. e U2OS cells were transfected with the indicated siRNAs 48 h before CldU (red) and IdU (green) labeling, followed by treatment with 4 mM HU for 5 h. The PARP inhibitor olaparib (10 μM) was optionally added 2 h before CldU addition. Track length analysis and statistics as in Fig. 3a
Fig. 4
Fig. 4
Stable RAD51 nucleofilaments are required not to form, but rather to protect reversed forks from nucleolytic degradation. a Control (BJ) or RAD51-T131P fibroblasts were labeled with CldU (red) and IdU (green), followed by treatment with 4 mM HU for 5 h and 50 μM mirin for 6 h, as indicated. A set of representative DNA fibers from each condition is shown. Ratios of IdU vs. CldU tracts are plotted. Track length analysis and statistics as in Fig. 3a. b EM-based assessment of the frequency of reversed replication forks isolated from BJ and T131P treated as indicated (4 mM HU for 5 h; 50 μM mirin for 6 h). In brackets, the total number of analyzed molecules. Results of two independent EM experiments are in Supplementary Table 7
Fig. 5
Fig. 5
RAD52 promotes stalled fork degradation in BRCA2-defective cells. a U2OS cells were transfected with siRNA before labeling (siBRCA2, 48 h; siRAD52, 24 h) with CldU (red) and IdU (green), followed by treatment with 4 mM HU for 5 h. The RAD52 inhibitor (AICAR 40 μM) was optionally added 2 h before CldU labeling. Left: levels of indicated proteins, assessed by western blot. TFIIH, loading control. Middle panel: a representative set of DNA fibers from each condition is shown. Right: ratios of IdU vs. CldU tracts are plotted. Track length analysis and statistics as in Fig. 3a. b EM-based assessment of the frequency of reversed replication forks isolated from U2OS cells transfected with control and siRNA against BRCA2 (48 h) and/or RAD52 (24 h). Cells were treated with 4 mM HU for 5 h and 50 μM mirin for 6 h, as indicated. In brackets, the total number of analyzed molecules. Results of two independent EM experiments are in Supplementary Table 8. c HEK293T cells were transfected by the indicated siRNAs 48 h before the EdU-labeling for 15 min and then treated with HU 4 mM for 5 h. AICAR 40 μM (RAD52 inhibitor) was optionally added 2 h before EdU labeling and retained throughout the experiment. Proteins associated with nascent DNA were isolated by iPOND (see “Methods” section) and detected with the indicated antibodies. For the thymidine chase experiment (Thy-chase), 10 μM thymidine was added for 2 h directly after the EdU labeling. In the control experiment (no EdU), the click reaction is performed using DMSO instead of biotin azide. The graph represents average and SD (error bars) of quantified MRE11 capture signals from three independent experiments. d Chromosomal breakage quantification after HU and mirin treatment (4 mM HU for 5 h; 50 μM mirin for 6 h) of U2OS cells after depletion of BRCA2 (48 h) and/or RAD52 (24 h). One hundred cells in pro-metaphase were analyzed. Similar results were obtained in two biological replicates
Fig. 6
Fig. 6
Fork reversal impairment suppresses fork degradation, but increases chromosomal breakage in BRCA2-defective cells. a Frequency of reversed replication forks isolated from unperturbed mouse ESCs—transfected with siLuc or siBrca2 (48 h)—and from Brca2−/− shPtip ESCs. In brackets, the total number of analyzed molecules. Results of two independent EM experiments are in Supplementary Table 9. b Representative count of chromatid breaks upon 5 h treatment with HU 4 mM in control and ZRANB3 knockout (KO) U2OS cells; where indicated, 50 μM mirin was added 1 h before HU treatment (6 h total treatment), and siRNA transfection was performed 48 h (BRCA2) or 20 h (PTIP) before HU treatment. The number of chromatid breaks per chromosome spread was plotted. At least 150 chromosome spreads were analyzed. Error bars represent SEM. A representative DAPI stained chromosome spread is shown. Insets 1 and 2 show intact chromosomes, while 3 and 4 display chromosomal breaks. c Chromosomal breakage quantification of HU-treated (4 mM HU, 5 h) U2OS cells after optional depletion of BRCA2 (48 h) and/or PARP inhibition (Olaparib 10 μM, added 2 h before HU). At least 180 cells in pro-metaphase were analyzed. The number of chromatid breaks per chromosome spread was plotted. Error bars represent SEM. Similar results were obtained in three biological replicates
Fig. 7
Fig. 7
Model for the role of different HR factors in stalled fork remodeling and protection. With the help of ZRANB3 and PARP activity, RAD51 promotes efficient reversal of stalled replication forks independently of BRCA2. Upon initial resection of reversed forks, RAD51 is efficiently loaded by BRCA2 on regressed arms to limit MRE11/PTIP/RAD52-dependent nucleolytic degradation and promote efficient fork restart. In BRCA2-defective cells, deregulated MRE11-dependent degradation of reversed forks leads to ssDNA accumulation and chromosomal breaks. Limiting reversed fork degradation restores fork integrity and prevents chromosomal breakage. Preventing fork reversal also restores fork integrity in BRCA2-defective cells—by reduced availability of degradation substrates—but leads to increased chromosomal breakage, and is thus detrimental for genome stability

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References

    1. Roy R, Chun J, Powell SN. BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat. Rev. Cancer. 2012;12:68–78. doi: 10.1038/nrc3181. - DOI - PMC - PubMed
    1. Thorslund T, et al. The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA. Nat. Struct. Mol. Biol. 2010;17:1263–1265. doi: 10.1038/nsmb.1905. - DOI - PMC - PubMed
    1. Lord CJ, Ashworth A. Mechanisms of resistance to therapies targeting BRCA-mutant cancers. Nat. Med. 2013;19:1381–1388. doi: 10.1038/nm.3369. - DOI - PubMed
    1. Schlacher K, et al. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell. 2011;145:529–542. doi: 10.1016/j.cell.2011.03.041. - DOI - PMC - PubMed
    1. Schlacher K, Wu H, Jasin M. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. Cancer Cell. 2012;22:106–116. doi: 10.1016/j.ccr.2012.05.015. - DOI - PMC - PubMed

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