The complex signaling pathways activated by genotoxins play important roles in maintaining genomic stability by blocking cell cycle progression, activating apoptosis, and influencing DNA repair. As such, deficiencies in checkpoint signaling can result in cancer-causing mutations that lead to tumorigenesis. Perhaps paradoxically, checkpoint signaling also facilitates the survival of tumor cells treated with genotoxic anticancer therapies, and efforts are currently underway to therapeutically target checkpoint signaling to increase chemotherapy effectiveness. One important checkpoint signaling pathway is the ATR-Chk1 pathway. Activation of this pathway requires Rad9, which forms a heterotrimeric complex with Hus1 and Rad1 (the 9-1-1 complex) that is loaded onto chromatin at sites of DNA damage and replication stress. Numerous single nucleotide polymorphisms (SNPs) have been identified in the coding region of the Rad9 gene, including SNPs that alter the amino acid sequence of Rad9 (known as nonsynonymous cSNPs). The effects of these cSNP on Rad9 function are currently unknown. Given that Rad9 is vital for checkpoint activation, understanding the biochemical mechanism of these cSNPs will determine how these polymorphisms affect checkpoint signaling. Therefore, in this proposal, I will undertake a comprehensive analysis of the known Rad9 cSNPs by assessing the following specific aims: 1. Do the nonsynonymous cSNPs of Rad9 affect the interaction of Rad9 with its binding partners? 2. Do Rad9 nonsynonymous cSNPs affect stress-induced checkpoint signaling in a model system? 3. Do nonsynonymous Rad9 cSNPs exert a dominant effect on checkpoint activation? Upon completion of these studies, I will have advanced our understanding of how Rad9 cSNPs impact activation of the ATR-Chk1 signaling pathway, a pathway that plays critical roles in the maintenance of genomic stability as well as in the responses of normal and tumor cells to genotoxic anticancer therapies. Taken together, my findings will lay the foundation for future studies that will address the potential impacts of Rad9 cSNPs on human health, including the development and treatment of neoplasms.
