Project 3: Antagonistic Mechanisms of BRCA2 and the 53BP1 Axis in RAD51 Nucleoprotein Filament Assembly

Project Summary Homology-directed repair (HDR) is a major, high-fidelity pathway for the elimination of DNA double-strand breaks (DSB)s and for DNA replication fork maintenance. Deficiencies in HDR invariably cause genome destabilization and can lead to neoplastic cell transformation and tumorigenesis. HDR is initiated via the exonucleolytic resection of the 5’ strand of DSBs to generate extended stretches of ssDNA. DNA resection is subject to negative control by 53BP1 and associated factors including DYNLL1 and the hetero-tetrameric Shieldin (SHLD1,2,3-REV7) and the CTC1-STN1-TEN1 (CST) heterotrimer. The mechanism of DNA end resection restriction by the 53BP1 complex and the manner in which this restrictive action is overcome by the tumor suppressor complex BRCA1-BARD1 will be defined in Project 1 and Project 2. In HDR, the 3’ ssDNA tails derived from DNA end resection serves as the template for the assembly of a helical nucleoprotein filament of the RAD51 recombinase. The assembly of the RAD51-ssDNA nucleoprotein filament is facilitated by the tumor suppressor BRCA2 in conjunction with its obligatory partner DSS1. The presynaptic filament catalyzes DNA strand invasion, a crucial step of HDR. Surprisingly, we have found that CST strongly inhibits the DNA strand exchange activity of the RAD51-ssDNA nucleoprotein filament. This result indicates a novel post-resection function of CST in regulation of DSB repair. Furthermore, we have found that the inhibitory effect of CST is alleviated by the BRCA2-DSS1 complex. These paradigm-shifting results provide new insights into the mechanism of DSB repair pathway choice. Project 3 will delineate the mechanistic underpinnings of the novel post-resection regulatory circuit of DSB repair in humans. Our aims are to study: 1) the mechanism of CST-imposed restriction of RAD51-promoted DNA strand exchange; 2) the mechanism of BRCA2-DSS1 reversal of CST-imposed restriction; and 3) the role of the higher order BRCA complexes in overcoming CST restriction. Because mutations in BRCA2 and BRCA1 proteins are responsible for nearly a half of all hereditary breast and ovarian cancers, the proposed studies will not only uncover the fundamental mechanisms of regulation of HDR in humans which involve these proteins but will also help in the development of novel cancer therapeutics.