Analysis of Eucaryotic DNA Polymerase

真核 DNA 聚合酶的分析

• 批准号: 9020121
• 负责人: Tim Herman
• 金额: $ 18.8万
• 依托单位: Medical College of Wisconsin
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9020121&HistoricalAwards=false
• 关键词: Analysis; Eucaryotic; DNA; Polymerase

The goal of this proposal is to investigate the interaction of a eucaryotic DNA polymerase with its primer-template. Our approach involves photo-crosslinking of oligonucleotide primer- templates to active site peptides of the multi-subunit DNA polymerase. The DNA primase activity of polymerase alpha and the 3' - 5' exonuclease active site of DNA polymerase epsilon will be investigated using modifications of this basic procedure. Two different classes of photoactive nucleotide analogs will be used in this work. In the first, (5-azido- dUTP, 8-azido-dATP and 8-azido-dGTP) the photoactive azido group is attached directly to the pyrimidine or purine base. The primary amino group of this deprotected oligonucleotide will then be reacted with a series of photoactive, heterobifunctional crosslinking reagents to generate oligonucleotide primers with specifically-positioned, cleavable, photoactive nucleotide analogs. The ultimate goal of this proposal is to isolate and identify by sequence analysis, specific active site tryptic peptides crosslinked to oligonucleotide primer-templates. By mapping the active sites of DNA polymerase first to specific subunits of the complex, and later to specific tryptic peptides within the subunit, we hope to begin to understand the structural and functional organization of this remarkable enzyme complex.

本提案的目的是研究以下因素的相互作用： 一种带有引物模板的真核DNA聚合酶。 我们 方法涉及寡核苷酸引物的光交联， 模板到多亚基DNA的活性位点肽 聚合酶。 聚合酶α的DNA引发酶活性和 DNA聚合酶3 ′-5 ′外切酶活性位点 将使用这种基本的修改进行研究 procedure. 两种不同类型的光敏核苷酸 在这项工作中将使用类似物。 在第一种情况下，（5-叠氮基- dUTP、8-叠氮基-dATP和8-叠氮基-dGTP）的光活性叠氮基 基团直接连接到嘧啶或嘌呤碱基上。 该去保护的寡核苷酸的伯氨基 然后会与一系列的光活性物质反应， 异双官能交联试剂， 具有特异性定位的寡核苷酸引物， 可裂解的光敏核苷酸类似物。 最终目标 这个建议的一个重要方面是通过序列分离和鉴定 分析，特异性活性位点胰蛋白酶肽交联， 寡核苷酸引物-模板。 通过绘制活性位点 DNA聚合酶首先作用于复合物的特定亚基， 然后是亚基内的特异性胰蛋白酶肽， 我希望开始去了解 这一非凡的酶复合体的组织。