Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection

Abstract DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by DNA end resection, a process involving the controlled degradation of the 5'-terminated strands at DSB sites 1,2. The breast cancer suppressor BRCA1-BARD1 not only promotes resection and HR, but it also protects DNA upon replication stress 1,3-9. While BRCA1-BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, its direct role in resection has been unclear, particularly due to complex phenotypes associated with the depletion of this essential and multi-functional complex 10-18. Using purified recombinant proteins, we show here that BRCA1-BARD1 directly promotes long-range DNA end resection pathways catalyzed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1-BARD1 stimulates DNA unwinding by the WRN/BLM RecQ family helicase. Together with MRE11-RAD50-NBS1 (MRN) and phosphorylated CtIP, BRCA1-BARD1 forms the BRCA1-C complex 19,20, which stimulates resection synergistically to even a greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1-C complex 21-23, inhibits resection, showing that BRCA1-C is a functionally integrated ensemble directly promoting long-range resection. While BRCA1-BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves an HR-independent function of the recombinase RAD51 4-6,8. We show that in the presence of RAD51, BRCA1-BARD1 instead inhibits DNA degradation. Based on our data, the presence and local concentration of RAD51 might determine the balance between the pro-nuclease and the DNA protection functions of BRCA1-BARD1 in the various physiological contexts.