NFBD1/MDC1, 53BP1 and BRCA1 have both redundant and unique roles in the ATM pathway

NFBD1/MDC1, 53BP1, and BRCA1 are DNA damage checkpoint proteins with twin BRCT domains. In order to determine if they have redundant roles in responses to ionizing radiation, we used siRNA and shRNA to deplete NFBD1, 53BP1, and BRCA1 in single, double, and triple combinations. These analyses were performed in early passage human foreskin fibroblasts so that checkpoint responses could be assessed in a normal genetic background. We report that NFBD1, 53BP1, and BRCA1 have both unique and redundant functions in radiation-induced phosphorylation and localization events in the ATM-Chk2 pathway. 53BP1, but not NFBD1 and BRCA1, mediates ionizing radiation-induced ATM S1981 autophosphorylation. In contrast, all three mediators collaborate to promote IR-induced Chk2 T68 phosphorylation. NFBD1 and 53BP1, but not BRCA1, work together to mediate pATMS1981, pChk2T68, and NBS1 ionizing radiation induced foci (IRIF). However, the relative importance of NFBD1 and 53BP1 in IRIF formation differ. We also determined the interdependence among mediators in IRIF recruitment. We extend previous findings in cancer cells and mouse cells that NFBD1 is upstream of 53BP1 and BRCA1 to primary human cells. Furthermore, NFBD1 promotes BRCA1 IRIF through both 53BP1-dependent and 53BP1-independent mechanisms.