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 chaperone-
like 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.