PROTEIN-DNA INTERACTIONS--AFFINITY LABELING APPROACH

蛋白质-DNA 相互作用——亲和标记方法

• 批准号: 3466411
• 负责人: MICHAEL B DOUGHTY
• 金额: $ 9.91万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-30 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466411
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA replication; affinity labeling; computer graphics /printing; enzyme substrate; nucleic acid chemical synthesis; nucleotide analog; protein structure; synthetic nucleotide

A site-specific labeling approach for systematic investigation of substrate binding and catalytic mechanisms employed by nucleic acid processing enzymes is outlined. Synthesis of nucleotide photoaffinity analogs-- 5-azidodeoxyuridine 1, 2- azidodeoxyadenosine 2, and 2-benzoyldeoxyadenosine 3 mono- (series A) and tri- (series B) phosphates and 5'-p-azidosulfonyl- benzoyldeoxyadenosine-- is investigated. Incorporation of photoaffinity analogs 1 and 2 into poly(dT)10 and poly(dA)10, respectively, by enzymic methodologies provides primer and exonuclease active site affinity labels. DNA polymerase I is employed as a test system to demonstrate the effectiveness of our approach in complimenting kinetic, crystallographic, mutagenesis, and FTNMR results in elucidating molecular contacts between polymerases and their substrates. Extensive molecular graphics and energy minimization techniques correlate affinity labeling results with a molecular description of nucleic acid processing-- DNA polymerization, processivity, and proofreading. These techniques will eventually allow elucidation of fundamental differences between eukaryotic, prokaryotic, and viral nucleic acid processing, and the design, based on first principles, of molecules of pharmacological interest which can specifically inhibit errant nucleic acid processing associated with human disease states.

用于系统调查的特定地点标记方法 核酸所采用的底物结合和催化机制 概述了加工酶。 核苷酸的合成 光亲和类似物-- 5-叠氮脱氧尿苷 1, 2- 叠氮脱氧腺苷 2 和 2-苯甲酰脱氧腺苷 3 单-（系列 A）和三（B系列）磷酸酯和5'-对叠氮磺酰基- 苯甲酰脱氧腺苷——已被研究。 纳入 将光亲和类似物1和2转化为聚(dT)10和聚(dA)10， 分别通过酶学方法提供引物和 核酸外切酶活性位点亲和力标签。 DNA聚合酶I是 用作测试系统来证明我们的有效性 补充动力学、晶体学、诱变的方法， 和 FTNMR 结果阐明了之间的分子接触 聚合酶及其底物。 广泛的分子图形和 能量最小化技术关联亲和标记结果 具有核酸加工的分子描述——DNA 聚合、持续性和校对。 这些技巧 最终将阐明根本差异 真核、原核和病毒核酸加工之间， 以及基于第一原理的分子设计 药理作用，可以特异性抑制错误 与人类疾病状态相关的核酸加工。