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. 2012 Jan;40(2):726-38.
doi: 10.1093/nar/gkr748. Epub 2011 Sep 27.

A dual role of BRCA1 in two distinct homologous recombination mediated repair in response to replication arrest

Zhihui Feng  1 , Junran Zhang
Affiliations

A dual role of BRCA1 in two distinct homologous recombination mediated repair in response to replication arrest

Zhihui Feng et al. Nucleic Acids Res. 2012 Jan.

Abstract

Homologous recombination (HR) is a major mechanism utilized to repair blockage of DNA replication forks. Here, we report that a sister chromatid exchange (SCE) generated by crossover-associated HR efficiently occurs in response to replication fork stalling before any measurable DNA double-strand breaks (DSBs). Interestingly, SCE produced by replication fork collapse following DNA DSBs creation is specifically suppressed by ATR, a central regulator of the replication checkpoint. BRCA1 depletion leads to decreased RPA2 phosphorylation (RPA2-P) following replication fork stalling but has no obvious effect on RPA2-P following replication fork collapse. Importantly, we found that BRCA1 promotes RAD51 recruitment and SCE induced by replication fork stalling independent of ATR. In contrast, BRCA1 depletion leads to a more profound defect in RAD51 recruitment and SCE induced by replication fork collapse when ATR is depleted. We concluded that BRCA1 plays a dual role in two distinct HR-mediated repair upon replication fork stalling and collapse. Our data established a molecular basis for the observation that defective BRCA1 leads to a high sensitivity to agents that cause replication blocks without being associated with DSBs, and also implicate a novel mechanism by which loss of cell cycle checkpoints promotes BRCA1-associated tumorigenesis via enhancing HR defect resulting from BRCA1 deficiency. VSports手机版.

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Figures

Figure 1.
Figure 1.
SCE occurs following stalled or collapsed DNA replication forks. (A) The Comet assay was conducted in MCF7 cells treated with 2 mM HU. At least 150 cells were analyzed for each treatment. Results were expressed by Olive moment. P-values were calculated by Student's t-test (*P < 0.01). (B) Representative SCEs. Arrows indicate the discontinuity in the staining pattern of the metaphase chromosomes due to SCEs. (C) The levels of SCE in MCF7 cells with or without HU treatment. In brief, cells treated with 2 mM HU for indicated periods of time were grown in the presence of bromodeoxyuridine (BrdU) for 50–60 h and mitotic cells were prepared according to a standard procedure (see ‘Materials and Methods’ section). Histograms show the frequency of SCE per 1000 chromosomes with at least 40–50 metaphase cells being counted. The data shown is the result from three independent experiments. P-values were calculated by Student's t-test (*P < 0.01).
Figure 2.
Figure 2.
ATR suppresses the SCE induced by replication fork collapse. (A) ATR knockdown by shRNA targeting ATR (ATRsh). Lysates were prepared from MCF7 cells after 72-h infection with ATRsh or control shRNA (consh). (B) The SCEs induced by collapsed DNA replication forks are suppressed by ATR. At 48-h after infection with ATRsh, MCF7 cells were used for SCE assay, which is performed as described in Figure 1C. The data shown is the result from three independent experiments. P-values were calculated by Student's t-test (*P < 0.01). (C) Chk1 phosphorylation (Chk1-P) and RPA2-P (anti-RPA2-p4/8 antibody) are increased in cells treated with 2 mM HU for 6-h. Actin was detected as a load control. (D) Representative metaphase prepared in MCF7 cells treated with 18-h HU following ATR depletion by ATRsh. The chromosomes with SCE(s) were indicated by arrows. (E) ATR-deficient cells show significant reductions in HR mediated by short tract gene conversion. HR induced by I-Sce-I was measured by dual-color flow cytometric detection of GFP-positive cells. The relative HR frequencies in cells depleted of ATR are shown in comparison to cells with intact ATR expression. Results are means from three independent experiments, with standard errors shown. P-values were calculated by Student's t-test (*P < 0.01).
Figure 3.
Figure 3.
The role of BRCA1 in promotion of SCE induced by replication fork collapse is enhanced by ATR depletion. (A) BRCA1 knockdown via shRNA targeting BRCA1 (BRCA1sh) in MCF7 cells. (B) The roles of BRCA1 in SCE upon replication fork stalling and collapse are differentially regulated by ATR depletion. The MCF7 cells were infected by consh, BRCA1sh, ATRsh or both of BRCA1sh and ATRsh. Forty-eight hours after infection, the cells were treated with 2 mM HU for indicated periods of time. The frequencies of SCE per 1000 chromosomes in cells treated with HU are demonstrated. At least 40–50 metaphase cells are counted per group. Results are means from three independent experiments, with standard errors shown. P-values were calculated by Student's t-test (*P < 0.01). (C) The histogram shows the fraction of metaphases with 1–9 versus 10–19 versus 20–29 versus ≥30 SCEs count.
Figure 4.
Figure 4.
BRCA1 and RAD51 proteins are localized at DNA ssDNA or DSBs region in response to stalled or collapsed DNA replication forks. (A and B). Kinetics of RAD51 (A) or BRCA1 (B) focus formation. The percentage of cells with more than 10 nuclear foci was calculated. In each experiment, 200 nuclei were counted per data point. Error bars indicate standard errors from three independent experiments (*P < 0.01). (C) BRCA1 and RAD51 colocalizes at the ssDNA region in response to replication fork stalling (left panel). The protocol for ssDNA detection has been described in a previous publication (12,51). Cells treated with 6-h HU were co-stained by anti-BrdU antibody and antibody against RAD51 or BRCA1. Representative foci images in response to 6-h HU treatment were shown. BRCA1 and RAD51 protein colocalizes at DSBs region in response to replication fork collapse (right panel). The cells co-stained with anti-γ-H2AX antibodies and antibodies against RAD51 or BRCA1. Representative foci images in response to 18-h HU treatment were shown.
Figure 5.
Figure 5.
The roles of BRCA1 in promoting RAD51 recruitment induced by replication fork collapse are enhanced by ATR depletion. (A) BRCA1 and Rad51 colocalizes in response to replication fork stalling and collapsed. (B) The roles of BRCA1 in Rad51 recruitment upon replication fork stalling and collapse are differentially regulated by ATR depletion. Exponentially growing MCF7 cells were first infected with both of BRCA1sh and ATRsh. Forty-eight hours after infection, the cells treated with HU were fixed at indicated time points. Then the fixed cells were detected for RAD51 by immunostaining. The data are from three independent experiments. Y-axis represents the percentage cell with positive foci. Cells were scored positive when 10 nuclear foci were visible. Results are means from three independent experiments, with standard errors shown. P-values were calculated by Student's t-test (*P < 0.01).
Figure 6.
Figure 6.
BRCA1 depletion leads to decreased RPA2-P protein levels following replication fork stalling but has no effect on RPA2 protein levels following replication fork collapse. (A) Immunoblot analysis of RPA2-P expression in MCF7 cells with or without BRCA1 depletion before and after 2 mM HU treatment for 6-h or 18-h. Proteins were visualized by immunoblot with anti-RPA2-p4/8, or anti-RPA2 antibody. (B) RPA2-P foci in cells with or without BRCA1 depletion. Fixed cells were stained by anti-RPA2-p4/8 antibody. Cells were scored positive when 10 nuclear foci were visible. Data were collected from three independent experiments, and the mean with standard errors was calculated. P-value was calculated by Student's t-test (P > 0.05). (C) CTIP was downregulated by shRNA targeting CTIP (CTIPsh). The CTIP mRNA was amplified by real-time quantitative PCR. mRNA CTIP in cells with or without CTIPsh infection were presented as a relative value compared to control cells infected with consh. Data were collected from three independent experiments. P-values were calculated by Student's t-test (*P < 0.01). (D) CTIP knockdown results in a more profound defect in SCE induced by 18-h HU treatment when ATR is depleted compared to cells with intact ATR expression. The SCE assays were performed as described in Figure 1C. Results are means from three independent experiments, with standard errors shown (*P < 0.01, #P < 0.05). P-values were calculated by Student's t-test.
Figure 7.
Figure 7.
BRCA1 knockdown leads to increased frequencies of chromosome aberrations following replication fork stalling. (A–C) Frequencies of chromatid breaks (A) and chromosome breaks (B) and radial structure (C) in MCF7 cells. For each sample 40–50 metaphases were analyzed. The data shown are from one of two independent experiments. (D)The representative metaphase spreads following 6-h HU treatment in MCF7 cells depleted of BRCA1 are shown. FISH using telomeric probe reveals the pink color. Chromosomes were stained with DAPI (blue).
Figure 8.
Figure 8.
A dual role of BRCA1 in two distinct HR mediated repairs in response to replication arrest. BRCA1 promotes ssDNA gap repair by HR independent of ATR. In addition, BRCA1 facilitates the repair of DNA DSBs following replication fork collapse, which is suppressed by ATR. BRCA1 perhaps functions with CTIP in promoting ssDNA resection at stalling replication forks. However, BRCA1 promotes HR following replication fork collapse via multiple mechanisms, in which BRCA1 acts as a HR mediator/comediator and also facilitates ssDNA resection along with CTIP.
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