Specific Aim 1. To dissect the mechanism of CST-imposed restriction of RAD51-promoted DNA strand exchange.
- Aim 1A. To determine the mechanism of RAD51 inhibition by CST
- Aim 1B. Identification of the CST attributes critical for RAD51 inhibition
Specific Aim 2. To determine the mechanism of BRCA2-DSS1 reversal of CST-imposed restriction.
- Aim 2A. The roles of BRCA2 functional attributes in alleviating CST-imposed restriction
- Aim 2B. To determine the role of DSS1 in DNA strand exchange restriction relief
- Aim 2C. Testing the CST mutants deficient in DSS1 interaction in human cells.
Specific Aim 3. To investigate the role of the higher-order BRCA complexes in overcoming CST restriction.
Abstract
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 the tumor suppressor complex BRCA1-BARD1 overcomes this restrictive action will be defined in Project 1 and Project 2. In HDR, the 3’ ssDNA tails derived from DNA end resection serve as the template for the assembly of a helical nucleoprotein filament of the RAD51 recombinase. The tumor suppressor BRCA2 facilitates the assembly of the RAD51-ssDNA nucleoprotein filament 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 the 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 DSB repair pathway choice mechanism. 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 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.
