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. 2013 Jan;33(2):387-95.
doi: 10.1128/MCB.00465-12. Epub 2012 Nov 12.

Rad51 paralog complexes BCDX2 and CX3 act at different stages in the BRCA1-BRCA2-dependent homologous recombination pathway

Jarin Chun  1 , Erika S Buechelmaier, Simon N Powell
Affiliations

Rad51 paralog complexes BCDX2 and CX3 act at different stages in the BRCA1-BRCA2-dependent homologous recombination pathway

Jarin Chun et al. Mol Cell Biol. 2013 Jan.

Abstract

The Rad51 paralogs are required for homologous recombination (HR) and the maintenance of genomic stability. The molecular mechanisms by which the five vertebrate Rad51 paralogs regulate HR and genomic integrity remain unclear. The Rad51 paralogs associate with one another in two distinct complexes: Rad51B-Rad51C-Rad51D-XRCC2 (BCDX2) and Rad51C-XRCC3 (CX3). We find that the BCDX2 and CX3 complexes act at different stages of the HR pathway. In response to DNA damage, the BCDX2 complex acts downstream of BRCA2 recruitment but upstream of Rad51 recruitment. In contrast, the CX3 complex acts downstream of Rad51 recruitment but still has a marked impact on the measured frequency of homologous recombination. Both complexes are epistatic with BRCA2 and synthetically lethal with Rad52. We conclude that human Rad51 paralogs facilitate BRCA2-Rad51-dependent homologous recombination at different stages in the pathway and function independently of Rad52.

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Figures

Fig 1
Fig 1
Rad51 paralog depletions lead to HR deficiency. (a) Immunoblot analysis showing siRNA depletion of individual Rad51 paralog proteins in MCF7 and U2OS cells. Levels of Rad51 are not affected by Rad51 paralog siRNA. An illustration of the BCDX2 and CX3 Rad51 paralog complexes is shown at the top of the panel. (b) I-SceI break-induced HR in MCF7 and U2OS cells treated with a nontarget control (NT) or the indicated Rad51 paralog siRNA: Rad51B (B), Rad51C (C), Rad51D (D), XRCC2 (X2), and XRCC3 (X3). Data represent the means and SD of the results of three independent experiments.*, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to NT control [Student's t test]).
Fig 2
Fig 2
Rad51 paralog complexes act downstream of BRCA1 and BRCA2 recruitment to damage foci. (a) Representative confocal images of MCF7 cells transiently transfected with NT, Rad51D, XRCC3, and Rad51C siRNA and stained for BRCA1 and BRCA2 at 5 h after exposure to 10 Gy of ionizing radiation (IR). Colocalization of BRCA1 and BRCA2 is shown in the adjacent merged images. (b) Quantification of BRCA1 and BRCA2 foci was obtained by counting at least 100 nuclei and scoring the presence of >5 foci as positive. Data represent the means and SD of the results of three independent experiments.
Fig 3
Fig 3
Rad51 paralog complexes function at different stages in the HR pathway. (a) Representative confocal images of MCF7 cells transiently transfected with the indicated siRNA and stained for BRCA1 and Rad51 or for BRCA2 and Rad51. Colocalization of BRCA1 and Rad51 and of BRCA2 and Rad51 is shown in the adjacent merged images. (b) Quantification of cells containing foci was obtained by counting at least 100 nuclei and scoring the presence of >5 foci as positive. Data represent the means and SD of the results of three independent experiments. *, P < 0.05 (compared to NT exposed to 10 Gy IR [Student's t test]).
Fig 4
Fig 4
Rad51 focus formation is not impaired in XRCC3−/− cells. (a) Immunoblot analysis validating parental HCT116 cells (P), XRCC3−/− cells (X3−/−), and complemented cells (X3−/−+X3). (b) Representative confocal images of Rad51 foci in parental HCT116, XRCC3−/−, and XRCC3−/−+XRCC3 cells treated with 0 or 10 Gy IR and incubated for 1.5 and 5 h. (c) Quantification of cells containing Rad51 foci was obtained by counting at least 100 nuclei and scoring the presence of >5 foci as positive. Data represent the means and SD of the results of three independent experiments.
Fig 5
Fig 5
Depletion of both Rad51 paralog complexes does not result in an additive decrease in Rad51 focus formation. (a) Immunoblot analysis showing Rad51 paralog depletions by siRNA in MCF7 cells. (b) Representative confocal images of MCF7 cells transiently transfected with siRNA for the NT control, Rad51D (D), or XRCC2 (X2) in combination with NT, Rad51C (C), or XRCC3 (X3) and stained for Rad51. (c) Quantification of cells containing Rad51 foci was obtained by counting at least 100 nuclei and scoring >5 foci as positive. Data represent the means and SD of the results of three independent experiments.
Fig 6
Fig 6
Rad51 paralogs are epistatic with the BRCA2 pathway of HR. (a and b) I-SceI break-induced HR in U2OS cells (a) and MCF7 cells (b) treated with individual Rad51 paralog siRNAs in combination with NT siRNA or BRCA2 siRNA. Note that two different siRNA oligonucleotides were used for each Rad51 paralog in U2OS cells: Rad51B (B_1, B_6), Rad51D (D_2, D_1), XRCC2 (X2_6, X2_2), Rad51C (C_6, C_7), and XRCC3 (X3_1, X3_2). (c) Plating efficiency of MCF7 cells treated with NT control alone (NT/NT), NT control with Rad51D siRNA (NT/Rad51D), NT control with BRCA2 siRNA (NT/BRCA2), or Rad51D siRNA with BRCA2 siRNA (Rad51D/BRCA2). (d) I-SceI break-induced HR in EUFA423 transiently transfected with vector control (VC) or BRCA2 in combination with NT or Rad51D siRNA. The adjacent panel shows a corresponding immunoblot demonstrating BRCA2 complementation and Rad51D depletion in EUFA432 cells. Data from panels a, b, c, and d represent the means and SD of the results of three independent experiments.*, P < 0.05 (compared to NT control [Student's t test]).
Fig 7
Fig 7
Rad51 paralogs are not epistatic with the Rad52 pathway of HR. (a) I-SceI break-induced HR in U2OS cells treated with individual Rad51 paralog siRNA in combination with NT siRNA or Rad52 siRNA. Note that two different siRNA oligonucleotides were used for each Rad51 paralog: Rad51B (B_1, B_6), Rad51D (D_2, D_1), XRCC2 (X2_6, X2_2), Rad51C (C_6, C_7), and XRCC3 (X3_1, X3_2). (b) Rad51 analysis of foci from MCF7 cells treated with NT control alone (NT/NT), NT with Rad52 siRNA (NT/R52), NT with Rad51D siRNA (NT/D), or Rad52 siRNA and Rad51D siRNA (R52/D). (c) Plating efficiency of MCF7 cells treated with NT, Rad51D siRNA, or XRCC3 siRNA in combination with NT or Rad52 siRNA. Data represent the means and SD of the results of three independent experiments. *, P < 0.05 (compared to NT/D or NT/X3 [Student's t test]).
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