DESIGN OF SELECTIVE INHIBITORS OF DNA POLYMERASES

DNA 聚合酶选择性抑制剂的设计

• 批准号: 3270655
• 负责人: George E Wright
• 金额: $ 22.44万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-12-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270655
• 关键词: DNA directed DNA polymerase; SDS polyacrylamide gel electrophoresis; X ray crystallography; active sites; adduct; adenosine triphosphate; chemical synthesis; deoxyribonucleoside triphosphate; deoxyribonucleotides; diphosphonate; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate analog; guanosine triphosphate; high performance liquid chromatography; ion exchange chromatography; nuclear magnetic resonance spectroscopy; nucleotide analog; polynucleotides; protein degradation; protein structure function; radiotracer; reversed phase chromatography; thymus; tissue /cell culture; virus protein

This work is devoted to the development of synthetic reagents for use as tools in the study of the structure and function of replicative DNA polymerases. A long term goal is to define amino acids in the active site region of DNA polymerases and the mechanism by which amino acids in the site effect incorporation of dNTPs at a primer terminus. A model enzyme for these studies is bacteriophage T4 DNA polymerase. A second goal is to clarify the relationship between eukaryotic DNA polymerases alpha, delta, epsilon, and to provide reagents to help define their roles in the substeps of DNA replication in vivo. This work will build upon previous results indicating that the synthetic nucleotides, N2-(p-n-butylphenyl) dGTP (BuPdGTP) and 2-(p-n-butylanilino (BuAdATP), are potent and selective inhibitors of DNA polymerase alpha and T4 DNA polymerase, and that carbonyldiphosphonate (COMDP) is a selective inhibitor of DNA polymerase de a. Four specific aims are proposed: development and characterization of selective inhibitors of calf thymus and HeLa cell DNA polymerases delta and epsilon; dissection of the mechanism by which the potent pol alpha inhibitor, BuPdGTP, inhibits and is incorporated by bacteriophage T4 DNA polymerase; synthesis and use of reactive, active site-directed nucleotides to label and dissect the active sites of DNA polymerases alpha and delta and T4 DNA polymerase, and; synthesis and demonstration of stable dNTP and primer analogs for use in X-ray crystallographic study of T4 DNA polymerase. Methods of organic synthesis are used to prepare synthetic nucleotides and phosphonates as inhibitors, substrates and potential irreversible labels for selected DNA polymerases. Ion exchange and reverse phase chromatography and multinuclear NMR spectroscopy are techniques used to support the synthetic work. DNA polymerase isolation and purification, enzyme assay by radioactive substrate incorporation and by polyacrylamide gel electrophoretic (PAGE) analysis of primer-extension reactions, and isolation by HPLC of modified primers will be used for biochemical work. Experiments involving protein degradation and chemical and spectroscopic analysis of labelled amino acids or peptides will also be employed.

这项工作致力于开发合成试剂，以用作 研究复制DNA的结构和功能的工具 聚合酶。 一个长期目标是定义活性部位的氨基酸 DNA聚合酶的区域和氨基酸中的机制 DNTP在引物末端的位点效应掺入。 模型酶 这些研究是噬菌体T4 DNA聚合酶。 第二个目标是 澄清真核DNA聚合酶α，三角洲， Epsilon，并提供试剂以帮助定义其在取代中的作用 体内DNA复制的。 这项工作将基于以前的结果 表明合成核苷酸N2-（p-n-丁基苯基）DGTP （bupdgtp）和2-（p-n-butylanilino（buadatp），有效且有选择性 DNA聚合酶α和T4 DNA聚合酶的抑制剂，并且 Carbonyldiphosphonate（COMDP）是DNA聚合酶DE的选择性抑制剂 一个。 提出了四个具体目标：开发和表征 小牛胸腺和HeLa细胞DNA聚合酶的选择性抑制剂，以及 Epsilon;解剖有效的pol alpha的机制 抑制剂BupDGTP，抑制并由噬菌体T4 DNA掺入 聚合酶；合成和使用反应性，主动位置定向核苷酸 标记和剖析DNA聚合酶alpha和delta的活性位点 和T4 DNA聚合酶，以及；稳定DNTP的合成和演示 用于T4 DNA X射线晶体学研究的底漆类似物 聚合酶。 有机合成方法用于制备合成核苷酸和 磷酸盐作为抑制剂，底物和潜在不可逆标签 用于选定的DNA聚合酶。 离子交换和反向阶段 色谱和多核NMR光谱是用于 支持合成工作。 DNA聚合酶分离和纯化， 通过放射性底物掺入和聚丙烯酰胺的酶测定 凝胶电泳（页）分析引发反应和 通过HPLC隔离修饰引物将用于生化工作。 涉及蛋白质降解以及化学和光谱的实验 也将采用对标记的氨基酸或肽的分析。