Figure 3. The MCPH1-BRCA2 interaction is required for localization of Rad51 at sites of DNA damage.

(A) MCPH1 colocalizes with Rad51 after DNA damage. 293T cells either not transfected (Control) or transfected with a Myc-tagged MCPH1 expression construct (WTMCPH1) were stained with anti-Rad51 (Red) and anti-Myc (Green) antibodies 3hr after exposure to 10Gy of radiation to visualize ionizing radiation induced nuclear foci. Likewise, 293T cells transfected with MCPH1 siRNA for 48hr were exposed to 10Gy of radiation and stained with anti-Rad51 (Red) and anti-53BP1 (Green) antibodies after 3hr to visualize depletion of Rad51 foci. (B) Cells transfected with plasmids encoding Myc-tagged MCPH1 deletion constructs were treated and evaluated for MCPH1 and BRCA2 foci by staining for Myc (Red) and BRCA2 (green). Separately, the same cells were evaluated for Rad51 and Myc-tagged MCPH1 foci with Myc (green) and Rad51 (Red). (C) 293T cells transfected with FLAG-tagged wildtype BRCA2 (FLAG-WT-BRCA2) or FLAG-tagged BRCA2-Δ328−351 deletion mutant were incubated for 48h, exposed to 10 Gy of radiation and stained after 3hr for FLAG (Red) and BRCA2, MCPH1, Rad51 or MDC1 (Green). (D) Lysates (WCL) from 293T cells transfected with Flag-tagged wildtype BRCA2 (WT-BRCA2) and the Flag-tagged BRCA2-Δ328−351 deletion construct were co-immunoprecipitated with anti-Rad51 antibody and immunoblotted with anti-FLAG antibody. Lysates from cells expressing wildtype (WT-MCPH1) and deletion mutants of MCPH1 were co-immunoprecipitated with anti-BRCA2 antibody and immunoblotted with Rad51 antibody and with anti-Myc antibody to verify MCPH1 expression. VU423 FANCD1 cells (FA-D1) and A913 423/2−33 FANCD1 cells reconstituted with BRCA2 (FAD1+BRCA2) were transfected with Myc-MCPH1, immunoprecipitated with anti-Rad51 antibody and immunoblotted with anti-Myc and anti-BRCA2 antibodies. Lysates were immunoblotted with anti-Rad51 antibody to verify Rad51 expression.