Double-stranded DNA breaks (DSBs) are the primary genotoxic lesion of ionizing radiation (IR). DSB induction appears to determine the efficacy of IR and other DNA metabolism-based anti-tumor drugs as cancer therapeutic agents. Homologous recombination (HR) is an important DSB repair pathway and essential for cellular radiation resistance and genome stability. HR defects lead to genomic instability, a hallmark in the etiology of cancer. The long-term goal is to elucidate the mechanisms of HR. We focus on the postsynaptic steps of DNA synthesis that determine fidelity of recombinational DNA repair. The Specific Aims are: (1) Determine the mechanisms of recombination-associated DNA synthesis. Yeast is an ideal model system to unravel the complexity of DNA synthesis during HR. Distinct DNA synthesis steps are invoked in recombination models, and at least four different DNA polymerases have already been implicated. We will delineate the precise role of DNA Pol? in HR using biochemical and molecular approaches (Subaim 1A). We have identified a novel role of Pol 4 in homology-directed repair and will define its specific function by a combination of biochemical and genetic approaches (Subaim 1C). The experiments will also address the key question whether TLS polymerases are directly involved in recombination-associated DNA synthesis. In Subaims 1B and D, we will validate the general significance of our findings using the well conserved human proteins. (2) Identify mechanisms for regulating recombination-associated DNA synthesis. Multiple lines of genetic evidence from several model systems suggest that recombination-associated DNA synthesis is regulated but the mechanisms involved are unknown. We have identified a novel role of the Sgs1-Top3-Rmi1 (human BLM- TOPO3?-RMI1/2) to specifically dissolve yeast Rad51-mediated D-loops. Contrary to expectation this function is independent of Sgs1 helicase activity. We will define this novel mechanism in Subaim 2A and determine whether it cooperates with mismatch repair to proofread recombination and reject strand invasions with mismatches in Subaim 2B. The novel paradigms will be validated in Subaim 2C with the corresponding and highly conserved human proteins.