Mechanistic and Structure-Function Studies of Human DNA Polymerase Lambda

人类 DNA 聚合酶 Lambda 的机理和结构功能研究

基本信息

批准号：
7672475
负责人：
Zucai Suo
金额：
$ 28.5万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-14 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7672475
关键词：
2-Aminopurine 5&apos -deoxyguanosine 5&apos -deoxyribose phosphate lyase Active Sites Affect Affinity Amino Acids Antibodies Antiviral Agents BRCA1 Protein BRCA1 gene Base Excision Repairs Binding Biochemical Biological C-terminal Canada Cell Line Chemistry Classification Clinical Collaborations DNA DNA Repair DNA Sequence Rearrangement DNA biosynthesis DNA lesion DNA polymerase beta2 DNA-Directed DNA Polymerase Data Deoxyuridine Embryo Enzymes Family Fibroblasts Fill-It Fluorescence Gene Rearrangement Generations Goals Homologous Gene Human Hydrogen Bonding IGH@ gene cluster Immunoglobulins In Situ In Vitro Individual Investigation Kinetics Length Lesion Letters Light Malignant Neoplasms Mammalian Cell Methods Molecular Molecular Conformation Mus N-terminal Nonhomologous DNA End Joining Nuclear Localization Signal Nucleotides Paper Pathway interactions Pharmaceutical Preparations Physiologic pulse Play Polymerase Predisposition Principal Investigator Proline-Rich Domain Property Protein Engineering Proteins Publishing Recruitment Activity Ribonucleotides Ribose Role Site Site-Directed Mutagenesis Structure Structure-Activity Relationship Techniques Thermodynamics Time Toxic effect United States Food and Drug Administration United States National Institutes of Health V(D)J Recombination Vertebral column X-Ray Crystallography antiviral nucleoside analog base drug efficacy human DNA in vivo inorganic phosphate insight malignant breast neoplasm novel nucleoside analog nucleotide analog oxidative damage polymerization research study sugar tripolyphosphate

项目摘要

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.

描述（申请人提供）：DNA 聚合酶 lambda (Pol;) 是最近鉴定的 X 家族 DNA 聚合酶，独特地包含 N 端核定位信号基序、乳腺癌易感蛋白 BRCA1 C 端 (BRCT) 结构域、富含脯氨酸的结构域和 C 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均缺乏催化活性，但可能影响 Pol; 的酶功能。最近，波尔；已被发现影响 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 损伤位点并进行原位修复。这些以及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充当间隙填充 DNA 聚合酶。主要研究者的长期目标是为 Pol 的间隙填充保真度、效率和持续性建立动力学、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。在本申请中，人类Pol；是具有以下具体目标的酶靶标：i) 确定 BRCT 和富含脯氨酸结构域对缺口填充 DNA 合成的影响，并评估这三个结构域和 Pol 的两种酶活性的细胞作用；; ii) 研究 DNA 和核苷酸的结构改变对核苷酸掺入动力学的影响，同时共同检查 FDA 批准的抗癌和抗病毒核苷类似物的功效和毒性； iii) 采用前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、前稳态动力学方法和 X 射线晶体学来建立 Pol; 中的结构-功能关系。我们的结果将提供人类 Pol 催化的缺口填充 DNA 合成的全面视图；并促进其生物学作用的识别。此外，我们的研究成果应该能够在分子水平上揭示免疫球蛋白的生成、DNA 修复和癌症形成。项目叙述通过对一种新型人类酶的研究，该项目旨在评估 FDA 批准的抗癌和抗病毒核苷类似物的功效和毒性，并在分子水平上了解抗体的生成、DNA 损伤修复和癌症形成。