No abstract

No abstract

Homologous recombinational repair (HRR) of DNA damage is critical for maintaining genome stability and tumor suppression. RAD51 and BRCA2 colocalization in nuclear foci is a hallmark of HRR. BRCA2 has important roles in RAD51 focus formation and HRR of DNA double-strand breaks (DSBs). We previously reported that BCCIP␣ interacts with BRCA2. We show that a second isoform, BCCIP␤, also interacts with BRCA2 and that this interaction occurs in a region shared by BCCIP␣ and BCCIP␤. We further show that chromatin-bound BRCA2 colocalizes with BCCIP nuclear foci and that most radiation-induced RAD51 foci colocalize with BCCIP. Reducing BCCIP␣ by 90% or BCCIP␤ by 50% by RNA interference markedly reduces RAD51 and BRCA2 foci and reduces HRR of DSBs by 20-to 100-fold. Similarly, reducing BRCA2 by 50% reduces RAD51 and BCCIP foci. These data indicate that BCCIP is critical for BRCA2-and RAD51-dependent responses to DNA damage and HRR.DNA double-strand breaks (DSBs) are induced by exogenous agents, such as ionizing radiation (IR), and arise spontaneously during normal DNA metabolism, such as at blocked or collapsed replication forks (9,10,39,45). Defects in DSB repair confer genome instability associated with tumorigenesis. In mammalian cells, DSBs are repaired by nonhomologous end-joining and by homologous recombinational repair (HRR) (60,62,65). RAD51 binds single-stranded DNA (ssDNA) to form nucleoprotein filaments that are essential for strand transfer during HRR (23,44,61,66). RAD51 is normally dispersed in the nucleus, but upon DNA damage induction, it redistributes to nuclear foci that are presumed sites of HRR (6,7,14,20,31,46). RAD51 foci have been shown to be associated with ssDNA regions after DNA damage (46). Several HRR proteins, including XRCC2, XRCC3, RAD51B, RAD51C, RAD51D, and BRCA2, are important for RAD51 focus formation (1,5,7,43,55,56).BRCA2 has nine RAD51 binding regions, including eight BRC repeats encoded by exon 11 and a distinct RAD51 binding region encoded by exon 27 (8, 33, 69). Expression of individual BRC repeats interferes with RAD51 focus formation and HRR (5, 53, 70), indicating that RAD51-BRCA2 interactions are important for both processes. The C-terminal half of BRCA2 has three regions that are structurally related to the ssDNA binding region of RPA and bind ssDNA in vitro, suggesting that ssDNA binding is also important for BRCA2 function in HRR (71). These ssDNA binding regions occur in a region called conserved domain IV (30,48,73) or the BRCA2 C-terminal domain (71), which is the longest and most evolutionarily conserved BRCA2 domain (32, 57). This domain also has binding sites for several proteins including DSS1, BUBR1, ABP-280/filamin-A, and BCCIP␣ (16,30,34,73).BCCIP␣ is a BRCA2 and CDKN1A (p21, Cip1, and Waf1) interaction protein (30); it has also been called . A second isoform, BCCIP␤, shares an N-terminal acidic domain and a central conserved domain but has a distinct C-terminal domain (Fig. 1A). In this report, BCCIP indicates both proteins. The BCCIP proteins share no significan...

BRCA1 plays a critical role in homology-directed repair (HDR) of DNA double strand breaks, and the repair defect of BRCA1-mutant cancer cells is being targeted with platinum drugs and poly (ADP-ribose) polymerase (PARP) inhibitors. We have employed relatively simple and sensitive assays to determine the function of BRCA1 variants or mutants in two HDR mechanisms, homologous recombination (HR) and single strand annealing (SSA), and in conferring resistance to cisplatin and olaparib in human cancer cells. Our results define the functionality of the top 22 patient-derived BRCA1 missense variants and the contribution of different domains of BRCA1 and its E3 ubiquitin ligase activity to HDR and drug resistance. Importantly, our results also demonstrate that the BRCA1-PALB2 interaction dictates the choice between HR and SSA. These studies establish functional and mutational landscapes of BRCA1 for HDR and therapy resistance, while revealing novel insights into BRCA1 regulatory mechanisms and HDR pathway choice.DOI: http://dx.doi.org/10.7554/eLife.21350.001