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. 2011 Jul;7(7):e1002148.
doi: 10.1371/journal.pgen.1002148. Epub 2011 Jul 14.

The Epistatic Relationship Between BRCA2 and the Other RAD51 Mediators in Homologous Recombination

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Free PMC article

The Epistatic Relationship Between BRCA2 and the Other RAD51 Mediators in Homologous Recombination

Yong Qing et al. PLoS Genet. .
Free PMC article

Abstract

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA-damage site.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Gene disruption of the SFR1, SWS1, and BRCA2 loci.
(A) Schematic representation of the endogenous SFR1 locus and gene-disruption constructs carrying the puro or bsr selection marker gene. The solid boxes represent exons, and numbers right above boxes represent exon numbers. Relevant BamHI and EcoRI sites are indicated. (B) Southern-blot analysis of genomic DNA digested by both BamHI and EcoRI was performed using the probe DNA shown in (A). Positions of hybridizing fragments of wild-type (WT) and targeted loci are indicated. (C) Schematic representation of the endogenous SWS1 locus and gene-disruption constructs carrying the puro or his selection marker gene. Relevant EcoRV and NotI sites are indicated. (D) Southern-blot analysis of genomic DNA digested by EcoRV and NotI was performed with the probe DNA shown in (C). (E) Experimental methods to generate BRCA2−/− and RAD51mediator−/−/BRCA2−/− cells. We generated BRCA2−/− cells from conditional mutant BRCA2−/con1 cells. In the minus allele of the BRCA2−/con1 cells, the whole coding sequence is deleted (hereafter called the coding sequence deletion allele). The structures of the conditional-null allele-1 (con1) is shown in (F). Treatment of BRCA2−/con1 cells with 4-OH tamoxifen (TAM) led to the generation of BRCA2−/− cells. To generate RAD51mediator−/−/BRCA2−/− cells, we disrupted one of the RAD51mediator genes in BRCA2−/con1 cells. Exposure of the resulting RAD51mediator−/−/BRCA2−/con1 cells to TAM led to the generation of RAD51mediator−/−/BRCA2−/− cells. (F) Schematic representation of BRCA2 conditional-null allele and the brca2-null allele wherein the whole coding sequences are deleted. The conditional-null allele-1 (con1) shown on top was described previously ; the structure of the coding sequence deletion allele is shown in the second row. Treatment of the BRCA2−/con1 cells with TAM causes deletion of the promoter and initiation codon. The relevant EcoRI sites in the conditional-null allele-1, the relevant XbaI sites in the coding sequence deletion allele, and the position of the probes used in the Southern-blot analysis (G) are indicated. The solid boxes and arrowheads represent the exons and loxP signals, respectively. (G) Southern-blot analysis of the conditional allele (left) and the other coding sequence deletion (−) allele (right) in BRCA2−/con1 cells with (+) or without (−) TAM treatment. Southern-blot analysis of EcoRI or XbaI-digested genomic DNA was performed with the probe DNA shown in (F). (H) Western-blot analysis to verify the loss of BRCA2 protein in BRCA2−/− cells derived from BRCA2−/con1 cells.
Figure 2
Figure 2. Decreased cellular proliferation in brca2-null cells.
(A) Growth curve for cells of the indicated genotype. (B) The relative rate of cell growth per 8 hours (a single cell cycle for wild-type cells) plotted for cells carrying the indicated genotypes. Each value represents the averaged results from three separate experiments. Error bars represent standard deviation. (C) Cell-cycle distribution of brca2-null cells that were pulse-labeled with BrdU for 10 minutes and subsequently stained with FITC-conjugated anti-BrdU antibody (Y-axis, log scale) and propidium iodide (PI) (X-axis, linear scale). The upper gate indicates cells incorporating BrdU (S phase), the lower middle gate indicates G1 cells, and the lower-right gate indicates G2/M cells. The sub G1 fraction (lower-left gate) indicates dead cells. The number in each gate indicates the percentage of gated events.
Figure 3
Figure 3. Cellular tolerance to camptothecin, cisplatin, and olaparib.
(A) Cells of the indicated genotype were exposed to camptothecin for 72 hours, a period during which wild-type cells are able to divide nine times in the absence of exogenous DNA damage. The X-axis represents the concentration of camptothecin and the Y-axis represents the relative number of surviving cells at 72 hours. The vertical dotted lines show LC50 values (the concentration of camptothecin that reduces cellular survival to 50% relative to cellular survival without camptothecin treatment). Relative LC50 values of camptothecin (B), cisplatin (C), and Poly(ADP-ribose) polymerase inhibitor olaparib (D and E) are shown. Values shown are mean ± SD.
Figure 4
Figure 4. γ-ray–induced Rad51 subnuclear foci in RAD51-mediator mutant cells.
(A) Immuno-staining of irradiated wild-type and mutant DT40 clones using anti-RAD51 antibody. Cells were fixed 3 hours after irradiation with 4Gy γ-rays. Bar, 10 µm. (B) Quantification of RAD51 foci in individual cells of the indicated genotype. Data shown are the means of three experiments. Error bars indicate standard deviation. Statistical analysis was performed using the t test. * P<0.01 compared to wild-type.
Figure 5
Figure 5. Effect of brca2 deletion on sws1-, sfr1-, rad52-, rad54-, xrcc3-, and brca1-deficient cells.
Cellular sensitivities to the indicated DNA damaging agents were analyzed using the same method as in Figure 3. The LC50 values are shown in Figure 3B–3E.
Figure 6
Figure 6. Model of BRCA2 dependent regulation of various RAD51 mediators at DNA–damage sites.
In the absence of BRCA2, RAD51 polymer assembles at DNA damage sites (top), while BRCA2 significantly promotes the polymerization of RAD51 (bottom). This promotion is attributable to the requirement of BRCA2 for the appropriate localization of other RAD51 mediators at DNA damage sites. Accordingly, the loss of BRCA2 may result in the abnormal functioning of some RAD51 mediators, for example, SWS1. This scenario explains why the loss of SWS1 increased cellular tolerance to camptothecin, cisplatin, and olaparib only when BRCA2 was absent (Figure 3).

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