DNA polymerase lambda (Pol;), a recently identified X-family DNA polymerase, uniquely contains an N-terminal nuclear localization signal motif, a breast cancer susceptibility protein BRCA1 C- terminal (BRCT) domain, a Proline-rich domain, and a C-terminal polymerase 2-like domain. While the polymerase 2-like domain possesses 5'-deoxyribose-5-phosphate lyase and DNA polymerase activities, both the BRCT and Proline-rich domains lack catalytic activities but may influence the enzymatic functions of Pol;. Very recently, Pol;has been found to affect immunoglobulin heavy chain gene rearrangement in Pol;-deficient mice, to protect mouse embryonic fibroblasts against oxidative damage, and to be recruited to sites of DNA damage and repair in situ. These and many other biochemical and biological data suggest that Pol;likely functions as a gap-filling DNA polymerase in V(D)J recombination, base excision repair, and non-homologous end-joining pathways. The long-term goals of the principal investigator are to establish kinetic, thermodynamic, and structural bases for the gap-filling fidelity, efficiency, and processivity of Pol;and to elucidate the role of its individual domains both in vitro and in vivo. In this application, human Pol;is the enzyme target with the following specific aims: i) determine the effect of the BRCT and Proline-rich domains on gap-filling DNA synthesis and evaluate the cellular role of the three domains and two enzymatic activities of Pol;;ii) investigate the effect of structural alterations in both DNA and nucleotide on the kinetics of nucleotide incorporation while co- examining the efficacy and toxicity of FDA-approved anticancer and antiviral nucleoside analogs;iii) elucidate the complete kinetic mechanism of nucleotide incorporation into single-nucleotide gapped DNA by employing pre-steady state kinetic methods;iv) employ site-directed mutagenesis, protein engineering, pre-steady state kinetic methods, and X-ray crystallography to establish the structure-function relationships in Pol;. Our results will provide a comprehensive view of the gap- filling DNA synthesis catalyzed by human Pol;and facilitate the identification of its biological roles. Furthermore, insights from our studies should shed light into immunoglobulin generation, DNA repair, and cancer formation at the molecular level. PROJECT NARRATIVE Through the investigation of a novel human enzyme, this project seeks to evaluate the efficacy and toxicity of FDA-approved anticancer and antiviral nucleoside analog and to understand antibody generation, DNA damage repair, and cancer formation at the molecular level.