PROTEIN-DNA INTERACTIONS--AFFINITY LABELING APPROACH

用于系统调查的特定地点标记法 核酸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)磷酸盐和5‘-对叠氮磺酰基- 对苯甲酰脱氧腺苷--进行了研究。成立为法团 光亲和类似物1和2形成聚(Dt)10和聚(Da)10， 分别由酶方法学提供了入门读物和 核酸外切酶活性位点亲和标记。DNA聚合酶I是 作为一种测试系统，以证明我们的 对动力学、结晶学、诱变、 和FT核磁共振结果阐明了分子间的接触 聚合酶及其底物。广泛的分子图形和 能量最小化技术将亲和标记结果关联起来 关于核酸加工的分子描述--DNA 聚合、加工性和校对。这些技术 最终将有助于阐明根本的区别 在真核、原核和病毒核酸处理之间， 基于第一性原理的分子的设计 能特异性抑制错误的药理作用 与人类疾病状态相关的核酸处理。

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.