Mechanistic and Structure-Function Studies of Human DNA Polymerase Lambda

描述(申请人提供)：DNA聚合酶lambda(Pol；)是最近发现的一种X家族DNA聚合酶，其独特的结构域包括N-末端的核定位信号基序、乳腺癌易感蛋白BRCA1的C-末端(BRCT)结构域、富含Pro的结构域和C-末端的聚合酶2样结构域。当聚合酶2结构域具有5‘-脱氧核糖-5-磷酸裂解酶和DNA聚合酶活性时，BRCT和富含Pro的结构域都缺乏催化活性，但可能影响POL的酶功能；最近，POL；被发现影响POL；缺陷小鼠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，并被招募到DNA损伤和原位修复的部位。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作为填补缺口的DNA聚合酶发挥作用。首席研究人员的长期目标是为POL填补缺口的保真度、效率和可加工性建立动力学、热力学和结构基础；并阐明其各个结构域在体外和体内的作用。在这一应用中，人的POL是具有以下特定目的的酶靶标：i)确定BRCT和富含Pro的结构域对填充缺口DNA合成的影响，并评价这三个结构域和POL的两种酶活性在细胞中的作用；ii)研究DNA和核苷酸的结构变化对核苷酸掺入动力学的影响，同时联合检测FDA批准的抗癌和抗病毒核苷类似物的有效性和毒性；iii)用稳态前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；利用定点突变、蛋白质工程、稳态前动力学方法和X射线结晶学建立POL的结构-功能关系；我们的结果将提供一个全面的视角，以填补缺口的DNA合成催化的人Pol；并有助于确定其生物学作用。此外，我们研究的洞察力应该会在分子水平上揭示免疫球蛋白的生成、DNA修复和癌症形成。 通过对一种新型人类酶的研究，该项目寻求评估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生成、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.