Mechanistic and Structure-Function Studies of Human DNA Polymerase Lambda

描述（由申请人提供）：DNA聚合酶lambda （Pol;）是最近发现的x家族DNA聚合酶，独特地包含一个n端核定位信号基序、一个乳腺癌易感蛋白BRCA1 C-末端（BRCT）结构域、一个富含脯氨酸的结构域和一个C-末端聚合酶2样结构域。聚合酶2样结构域具有5'-脱氧核糖-5-磷酸裂解酶和DNA聚合酶活性，而BRCT和富含脯氨酸的结构域都缺乏催化活性，但可能影响Pol的酶促功能。最近，波尔；已发现影响Pol免疫球蛋白重链基因重排；-缺陷小鼠，以保护小鼠胚胎成纤维细胞免受氧化损伤，并被招募到DNA损伤部位并原位修复。这些和许多其他生化和生物学数据表明，Pol；可能在V(D)J重组、碱基切除修复和非同源末端连接途径中起缺口填充DNA聚合酶的作用。首席研究员的长期目标是建立动力学、热力学和结构基础，以保证Pol填充空隙的保真度、效率和加工能力；并阐明其个体结构域在体外和体内的作用。在这个应用中，人类Pol；是酶靶标，具有以下具体目的：i)确定BRCT和富含脯氨酸的结构域对缺口填充DNA合成的影响，并评估Pol的三个结构域和两种酶活性在细胞中的作用；ii)研究DNA和核苷酸结构改变对核苷酸结合动力学的影响，同时共同研究fda批准的抗癌和抗病毒核苷类似物的疗效和毒性；iii)利用预稳态动力学方法阐明核苷酸进入单核苷酸缺口DNA的完整动力学机制；iv)利用定点诱变、蛋白质工程、稳态前动力学方法和x射线晶体学建立Pol的结构-功能关系；我们的研究结果将为人类Pol催化的补隙DNA合成提供一个全面的视角；并有助于识别其生物学作用。此外，从我们的研究中获得的见解应该在分子水平上阐明免疫球蛋白的产生、DNA修复和癌症的形成。

DESCRIPTION (provided by applicant): 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.