MCF-7 cells were treated with IR, stained for 纬H2AX, MDC1, RNF8, RNF168, 53BP1, Abraxas (CCDC98), BRCA1, BRCC36, Merit40 (NBA1) and RAP80, and then imaged using high-resolution three-dimensional (3-D) confocal microscopy to assess the relative localization of proteins at foci.
All BRCA1-A complex components displayed strong co-localization, which overlapped significantly with RNF8 and RNF168, but not with 纬H2AX and MDC1. Intriguingly, 53BP1 co-located well with 纬H2AX and MDC1, but remained separate from RNF8 and RNF168. These co-localization patterns were consistent for at least 3 h after IR.
The foci formations of 纬H2AX-MDC1-53BP1 and RNF8-RNF168-BRCA1-A complexes are spatially independent. Such divergence was not anticipated from prior studies on the recruitment of these proteins to foci. This information indicates that individual foci may represent distinct sites of DNA repair facilitated by a specific subset of DDR proteins.