Defects in cellular DMA metabolism have a direct role in many human disease processes. Impaired responses to DNA damage and defects in basal DNA repair have been implicated as causal factors in life- shortening diseases including cancer, trinucleotide repeat diseases such as Fragile X and Huntington's diseases, and genetic diseases such as Xeroderma Pigmentosum, Cockayne's and Werner's syndromes. Understanding the molecular mechanisms of cellular DNA replication and repair is essential for developing new treatments for these diseases. Replication protein A (RPA) is a multi-functional, single-stranded DMA-binding protein composed of subunits of 70-, 32- and 14-kDa. RPA is essential for DNA replication, DNA repair, recombination and coordination of the cellular response to DNA damage. RPA is phosphorylated on the N-terminus of 32-kDa subunit during S-phase and in response to DNA damage. Recent data demonstrate that RPA hyper- phosphorylation modulates RPA activity and plays a role in the cellular response to DNA damage. The mechanism of regulation of RPA is currently poorly understood. We hypothesize that hyper-phosphorylation of RPA induces a novel conformational change in RPA that alters specific RPA-protein and/or RPA-DNA interactions to regulate DNA metabolism. Studies proposed will test this hypothesis using a combination of biochemical, genetic and structural approaches to elucidate the mechanism of RPA regulation. In three independent Aims, we will: (1) define RPA-DNA interactions modulated by hyper-phosphorylation; (2) define RPA-proteininteractions modulated by hyper- phosphorylation and characterize changes in RPA activity in vitro and in vivo caused by hyper- phosphorylation; and (3) carry out structural analysis to define hyper-phosphorylation induced conformational changes in RPA. These studies will both elucidate the mechanism of the RPA regulatory switch and provide insights into RPA function in cellular DNA metabolism.