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：
7907662
负责人：
Zucai Suo
金额：
$ 28.22万
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 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7907662
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 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（由申请人提供）：DNA聚合酶lambda（POL;）是一种最近确定的X-家庭DNA聚合酶，独特地包含N端核定位信号基序，这是一种乳腺癌易感性蛋白BRCA1 C-末端（BRCT），是一个富含富含的含量的结构域，是一个富含富含的含量的结构域，是富含富含的含量的结构域，一个富含富含的域，一个富含含量的结构域，一个C- Term-Term-Terminal-terminal-terminal-terminal-terminal-terminal-terminal-terminal Polymerase 2-light insabase 2-lighl sighs sighly。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和富含脯氨酸的域都缺乏催化活性，但可能会影响Pol的酶促功能。最近，Pol；已发现在POL中影响免疫球蛋白重链基因重排；缺乏小鼠，以保护小鼠胚胎成纤维细胞免受氧化损伤，并将招募到DNA损伤的部位和原位修复。这些以及许多其他生化和生物学数据表明POL；在V（d）J重组，碱基切除修复和非同源最终连接途径中，可能是填充间隙的DNA聚合酶。主要研究者的长期目标是建立动力学，热力学和结构碱基，以填充POL的间隙填充，效率和加工性；并阐明其在体外和体内的各个领域的作用。在此应用中，人类pol；是具有以下特定目的的酶靶标：i）确定BRCT和富含脯氨酸的域对填充间隙填充DNA合成的影响，并评估三个结构域的细胞作用以及POL的两个酶活性； ii）研究了DNA和核苷酸中结构改变对核苷酸掺入动力学的影响，同时研究了FDA批准的抗癌和抗病毒核苷类似物的疗效和毒性； iii）通过采用稳态的状态动力学方法，阐明了核苷酸掺入到单核苷酸间隙DNA中的完整动力学机制； iv）采用位置定向的诱变，蛋白质工程，稳态的状态动力学方法和X射线晶体学来建立POL中的结构功能关系；我们的结果将为人类POL催化的间隙填充DNA合成提供全面的看法；并促进其生物学作用的识别。此外，我们研究的见解应阐明分子水平的免疫球蛋白产生，DNA修复和癌症形成。该项目通过研究一种新型人类酶进行叙述，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，并了解分子水平的抗体产生，DNA损伤修复以及癌症形成。