Mechanistic and Structure-Function Studies of Human DNA Polymerase Lambda

描述（由申请人提供）：DNA聚合酶λ（Pol;），一种最近鉴定的X家族DNA聚合酶，独特地含有N-末端核定位信号基序、乳腺癌易感蛋白BRCA 1 C-末端（BRCT）结构域、富含脯氨酸的结构域和C-末端聚合酶2样结构域。虽然聚合酶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，但BRCT和富含脯氨酸的结构域都缺乏催化活性，但可能影响Pol的酶功能。最近，Pol已发现其影响Pol缺陷小鼠中的免疫球蛋白重链基因重排，保护小鼠胚胎成纤维细胞免受氧化损伤，并被募集到DNA损伤位点和原位修复。这些和许多其他生物化学和生物学数据表明，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充当间隙填充DNA聚合酶。主要研究者的长期目标是建立动力学，热力学和结构基础的缺口填充保真度，效率和聚合物的持续合成能力;并阐明其各个领域在体外和体内的作用。i）确定BRCT和富含脯氨酸的结构域对间隙填充DNA合成的影响，并评估Pol的三个结构域和两种酶活性的细胞作用; ii）研究DNA和核苷酸两者中的结构改变对核苷酸掺入动力学的影响，同时共-检验FDA批准的抗癌和抗病毒核苷类似物的功效和毒性; iii）通过采用预稳态动力学方法阐明核苷酸掺入单核苷酸缺口DNA的完整动力学机制; iv）采用定点诱变、蛋白质工程、预稳态动力学方法和X射线晶体学来建立Pol中的结构-功能关系;我们的研究结果将提供一个全面的看法差距填补DNA合成催化人Pol;并促进其生物学功能的鉴定。此外，我们研究的见解应该在分子水平上阐明免疫球蛋白生成，DNA修复和癌症形成。 通过对一种新型人类酶的研究，本项目旨在评估FDA批准的抗癌和抗病毒核苷类似物的疗效和毒性，并在分子水平上了解抗体产生，DNA损伤修复和癌症形成。

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.