Molecular Mechanisms of Human DNA Polymerase B Catalysis, Fidelity and Selective

人类 DNA 聚合酶 B 催化、保真度和选择性的分子机制

• 批准号: 8591712
• 负责人: MYRON GOODMAN
• 金额: $ 58.05万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-03 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8591712
• 关键词: Active Sites; Catalysis; Cell-Free System; Chemicals; DNA-Directed DNA Polymerase; Electronics; Electrostatics; Family; Grant; Human; Instruction; Kinetics; Left; Malignant Neoplasms; Measurement; Molecular; Oxygen; Polymerase; Property; Research; analog; base; bisphosphonate; cancer cell; human DNA; innovation; member; mutant; repair enzyme; research study; theories; tool

DNA polymerase mechanisms of base selection and catalysis are explored using a tool-kit of dNTP analogs that have bisphosphonates in place of the B,Y-bridge oxygen. These analogs are Pol B substrates that have leaving groups with widely dispirate electronic properties, pKa4 values ranging from 7.8 to 12.3, enabling us to use presteady state kinetic measurements to determine the selection of right and wrong deoxynucleotides occurring at the chemical transition state. Especially important members of the toolkit include all four individually synthesized (R)- and (S)-B,Y-CHF and B,Y-CHCI diastereomers. Our recent observation of a pronounced stereoselection for (R)-CHF in Pol B involving an electrostatic interaction of F with Arg183, unique to family X pols such as Pol B, serves as the impetus for a

使用 dNTP 类似物工具包探索 DNA 聚合酶的碱基选择和催化机制，该类似物用双磷酸盐代替 B,Y 桥氧。这些类似物是 Pol B 底物，其离去基团具有广泛不同的电子特性，pKa4 值范围为 7.8 至 12.3，使我们能够使用稳态前动力学测量来确定在化学过渡态发生的正确和错误脱氧核苷酸的选择。该工具包特别重要的成员包括所有四种单独合成的 (R)-和 (S)-B,Y-CHF 和 B,Y-CHCl 非对映体。我们最近观察到 Pol B 中 (R)-CHF 的明显立体选择涉及 F 与 Arg183 的静电相互作用，这是 X 族 pol（例如 Pol B）所独有的，这推动了