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PROTEIN-DNA INTERACTIONS--AFFINITY LABELING APPROACH

蛋白质-DNA 相互作用——亲和标记方法

基本信息

批准号：
3466412
负责人：
MICHAEL B DOUGHTY
金额：
$ 10.33万
依托单位：
UNIVERSITY OF KANSAS LAWRENCE
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-09-30 至 1994-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3466412
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

项目摘要

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.

一种用于系统调查的特定位点标记方法底物结合和核酸催化机制加工酶概述。核苷酸合成光亲和类似物--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-（系列B）磷酸酯和5 ′-对叠氮磺酰基- 苯甲酰基脱氧腺苷。掺入光亲和类似物1和2转化为聚（dT）10和聚（dA）10，分别通过酶法提供引物和核酸外切酶活性位点亲和标记。 DNA聚合酶I是作为一个测试系统，以证明我们的有效性，补充动力学、晶体学、诱变和FTNMR结果阐明了分子之间的接触，聚合酶及其底物。广泛的分子图形和能量最小化技术关联亲和标记结果对核酸加工过程的分子描述-- DNA 聚合、持续性和校对。这些技术将最终阐明在真核、原核和病毒核酸加工之间，以及基于第一性原理的分子设计药理学兴趣，可以特异性抑制错误的与人类疾病状态相关的核酸加工。