Role of nuclear chaperones in genomic instability and carcinogenesis

SUMMARY Lesions, breaks, and errors in DNA are drivers of genomic instability resulting in a variety of cancers. The DNA damage response (DDR) is a signaling cascade that responds to breaks in the DNA and utilizes an array of DNA repair factors to correct the error and preserve genomic integrity. Cellular responses to DDR involve regulation of the cell cycle and signaling processes that either trigger DNA repair or programmed cell death. Proteins that function in DDR are shuttled into the nucleus in response to DNA damage. My PhD thesis work and the proposed goals center around understanding the mechanisms that regulate nuclear-cytoplasmic localization of DDR proteins. The F99 part of the proposal focuses on the regulation of Replication Protein A (RPA) by chaperone-like proteins. RPA is an essential single-stranded DNA (ssDNA) binding DDR factor that regulates all aspects of DNA metabolism including DNA replication, repair, and recombination. RPA is transported into the nucleus, recognizes, and binds ssDNA, and activates DDR by interacting with over three dozen RPA-interacting proteins (RIPs). How spurious RPA-RIP interactions are prevented in the cell in the absence of ssDNA has been a long-standing mystery. I have uncovered that Rtt105 (Regulator of Ty1 transposition 105), a chaperone-like protein, functions as a regulator by interacting with multiple domains of RPA and conformationally restraining the complex. This serves as an inhibitor of RPA-RIP interactions. Using sophisticated biophysical, biochemical, and structural tools I show that ssDNA binds to the RPA-Rtt105 complex and removes the restraints to promote recruitment of DDR factors. In higher eukaryotes, a protein called RPAIN (RPA-interacting protein) serves as the functional ortholog of Rtt105 and I will focus on deciphering its mechanism of action. In addition, using cryoEM, I will determine the structures of RPA bound to these chaperonelike proteins. In the K00 part of the proposal, I will focus on identifying chaperone-like proteins specific to other cancer-related DDR proteins such as BRCA1, BRCA2, RAD52, and PALB2. In addition, I will investigate the regulatory and signaling mechanisms that control nuclear-cytoplasmic distribution of DDR factors during DNA damage. Finally, using knowledge obtained from the biochemical and cellular studies I will develop targeted small molecule cancer therapeutic inhibitors to regulate DDR. The combined F99 and K00 training phases will provide me with the necessary skills towards an independent research career focused on generating targeted cancer therapeutics.