E. COLI SINGLE-STRANDED DNA BINDING PROTEIN

大肠杆菌单链 DNA 结合蛋白

• 批准号: 3286141
• 负责人: JOHN W CHASE
• 金额: $ 13.15万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286141
• 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

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中起着核心作用 复制、重组和修复。一般说来，这些蛋白质结合 与单链DNA紧密结合，并且没有已知的催化特性 他们自己的。来自大肠杆菌的SSB就是这样一种特殊的蛋白质 人们的兴趣和重要性在很大程度上是由于 基本的DNA酶学知识和执行详细操作的能力 该系统中的遗传分析。在这方面，E.Coli系统 唯一有资格作为相应的机械分析的模型 来自高等有机体的蛋白质。SSB基本上是一种简单的蛋白质，是一种 很多人都知道它的物理化学性质和基因 由于其缺陷而产生的作用以及某些体外实验 生化反应，特别是DNA复制，在其中它 参与其中。然而，尽管有这样的知识，我们并不理解 任何详细的生化反应都能感觉到这种蛋白质的作用机制。 其欺骗性的简单与其在各个方面的亲密参与形成了鲜明对比 DNA新陈代谢。因此，至关重要的是，我们 了解这种蛋白质和类似蛋白质的实际功能，并为此 我们将进行以下研究。单晶和单晶的晶体结构 某些SSB的变种将被确定。基因突变抑制了 将对SSB突变株的表型进行研究，以确定 其他蛋白质的改变可能会弥补SSB的缺陷。我们 将研究几种SSB样蛋白的体内和体外性质 从最近发现的可传播的质粒中分离出来。 将对SSB的单抗进行研究。SSB和RecA亲和力 柱子将被用来尝试识别可能相互作用的蛋白质 并具体研究SSB和recA之间的相互作用 蛋白。进一步试图阐明SSB、RecA和 通过研究单链DNA对某些特定基因的影响，将制成单链DNA RecA和ssDNA的反应(例如，D-环的形成)。最后，这是我们的 目的利用所有这些研究的综合信息来 合理设计野生型大肠杆菌SSB变异体，可用于 测试关于SSB功能域的各种想法及其 参与蛋白质的生化作用机制。