A comparison of BRCA1 nuclear localization with 14 DNA damage response proteins and domains: Identification of specific differences between BRCA1 and 53BP1 at DNA damage-induced foci

BRCA1 is an important mediator of the DNA damage response pathway. Previous studies have identified a number of proteins that associate with BRCA1 at nuclear foci after ionizing radiation (IR)-induced DNA damage. However, the co-localization patterns of BRCA1 and various DNA damage response proteins have not yet been systematically quantified and compared within the same experimental system. In this study, a new inducible human cell line was established to allow unambiguous detection of YFP-BRCA1 at nuclear foci. Quantitative 2-D microscopic analysis was performed to compare the intranuclear co-localization of YFP-BRCA1 with 10 cellular proteins and 4 cellular domains before and after IR. Intriguingly, YFP-BRCA1 displayed significantly better focal co-localization with BARD1, RAP80 and Abraxas than with the upstream foci-initiating proteins gamma H2AX and MDC1. In contrast to previous reports, we found that the co-localization between YFP-BRCA1 and 53BP1 foci was surprisingly weak. Quantitative analyses of 3-D confocal images showed that similar to 60% of 53BP1 foci were unrelated to YFP-BRCA1 foci, similar to 35% of foci were abutting and only similar to 5% of foci co-localized. The YFP-BRCA1 and 53BP1 nuclear foci were distinctively separated within the first 3 h after IR. In addition, in situ nuclear retention analysis revealed YFP-BRCA1 and BARD1 are less mobile than 53BP1 at IR-incluced nuclear foci. Our findings indicate that BRCA1-BARD1 and 53BP1 are proximal but not overlapping at DNA break sites and are consistent with recent evidence for distinct roles of these proteins in the DNA damage response pathway. (C) 2009 Elsevier Inc. All rights reserved.