Project Summary DNA double-strand break (DSB) is one of the most toxic DNA lesions in cells. DSB causes immediate chromosome breakage and must be repaired before chromosome segregation. Unfaithful repair of DSB may lead to genome rearrangements and ultimately tumorigenesis. Research in my laboratory strives to define the mechanism and regulation of DSB repair where homologous recombination (HR) serves as a major repair means. Eukaryotic single-stranded DNA binding protein, RPA, as a universal DNA replication and repair factor, is involved in multiple stages of the HR pathway including the DSB end resection by the Sgs1-Dna2 helicase/nuclease ensemble. Our recent work discovered RPA as a processive unit for Dna2- catalyzed ssDNA digestion, which provided a novel perspective on the regulation of ssDNA accessibility by RPA and inspired us to explore how RPA may serve as an integral component of the Sgs1-Dna2 machinery and coordinate their actions. Unexpectedly, our work on the Dna2- RPA ensemble revealed a novel role of Dna2 in the processing of an undefined intermediate in DSB repair likely derived from DNA repair synthesis and led us to discover an RPA mutant that constitutively dimerizes, which may serve as a powerful tool to understand the function of RPA dimerization. Both directions will also be explored in this proposal. Given the conservation of the HR pathway between yeast and humans, our work will shed light on the mechanism of DSB repair not only in yeast but also in human cells.