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E COLI SINGLE-STRANDED DNA BUILDING PROTEIN

大肠杆菌单链 DNA 构建蛋白

基本信息

批准号：
3286140
负责人：
JOHN W CHASE
金额：
$ 18.57万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
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：
1988-08-01 至 1991-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3286140
关键词：
DNA binding protein DNA repair Escherichia coli affinity chromatography bacterial DNA bacterial genetics bacterial proteins binding proteins gene expression monoclonal antibody nucleic acid sequence plasmids structural genes

项目摘要

Single-stranded DNA binding proteins are of central importance in DNA replication, recombination and repair. In general, these proteins bind tightly to single-stranded DNA and have no known catalytic properties of their own. SSB from E. coli is one such protein that is of particular interest and importance due in large measure to the advanced state of knowledge of basic DNA enzymology and the ability to perform detailed genetic analyses in that system. In this regard the E. coli system uniquely qualifies as a model for the mechanistic analysis of corresponding proteins from higher organisms. SSB is a basically simple protein and a great deal is known about its physicochemical properties and the genetic effects which result from its deficiency as well as certain in vitro biochemical reactions, particularly DNA replication, in which it participates. In spite of this knowledge, however, we do not understand in any detailed biochemical sense the mechanism of action of this protein. Its deceptive simplicity contrasts its intimate involvement in every aspect of DNA metabolism. It is, therefore, of basic importance that we understand how this and similar proteins actually function and to this end we will perform the following studies. The crystal structure of SSB and certain variant SSB's will be determined. Mutations which suppress the phenotype of ssb mutant strains will be studied in order to determine if alterations in other proteins might compensate for a deficiency in SSB. We will study in vivo and in vitro properties of several SSB-like proteins from transmissible plasmids which have recently been identified. Monoclonal antibodies to SSB will be studied. Both SSB and RecA affinity columns will be used to attempt to identify proteins which may interact with SSB and to specifically investigate the interaction of SSB and recA protein. Further attempts to elucidate interactions of SSB, RecA and single-stranded DNA will be made by studying the effect SSB has on certain reactions of RecA and ssDNA (e.g., D-loop formation). Finally, it is our aim to utilize the combined information from all of these studies to logically design variants of wild type E. coli SSB which can be used to test various ideas about the functional domains of SSB and their involvement in the biochemical mechanism of action of the protein.

单链DNA结合蛋白在DNA中至关重要复制重组和修复一般来说，这些蛋白质与单链DNA紧密结合，没有已知的催化特性，自己的. SSB来自E.大肠杆菌就是这样一种蛋白质，在很大程度上由于先进的国家的利益和重要性，基本的DNA酶学知识和执行详细在这个系统中进行基因分析。在这方面，E.杆菌系统唯一有资格作为一个模型的机制分析，来自高等生物的蛋白质 SSB基本上是一种简单的蛋白质和一种关于它的物理化学性质和遗传特性，由于其缺乏以及某些体外生化反应，特别是DNA复制，其中它参与。尽管有这些知识，但是，我们不明白，任何详细的生化意义上的作用机制，这种蛋白质。其欺骗性的简单性与其在各个方面的密切参与形成了鲜明对比 DNA代谢。因此，至关重要的是，了解这种蛋白质和类似蛋白质的实际功能，我们会进行以下研究。 SSB的晶体结构和将确定某些变体SSB。突变抑制了将研究SSB突变株的表型，以确定是否其他蛋白质的改变可以弥补SSB的缺陷。我们将研究几种SSB样蛋白的体内和体外特性最近被鉴定出的可传播质粒。将研究抗SSB的单克隆抗体。 SSB和RecA亲和力将使用色谱柱来尝试鉴定可能相互作用的蛋白质与SSB，并专门研究SSB和recA的相互作用蛋白进一步尝试阐明SSB、RecA和单链DNA将通过研究SSB对某些 RecA和ssDNA的反应（例如，D环形成）。最后，我们的旨在利用所有这些研究的综合信息，野生型E的逻辑设计变体。coli SSB，可用于测试关于SSB功能域及其参与蛋白质的生化作用机制。