Structural Studies of RecA-DNA Complexes

RecA-DNA 复合物的结构研究

• 批准号: 6891303
• 负责人: CHARLES E BELL
• 金额: $ 25.81万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891303
• 关键词: DNA; DNA repair; DNA replication; Escherichia coli; SDS polyacrylamide gel electrophoresis; X ray crystallography; adenosinetriphosphatase; bacterial proteins; binding sites; enzyme complex; gene targeting; high performance liquid chromatography; mass spectrometry; nucleic acid metabolism; nucleic acid sequence; nucleic acid structure; protein structure function; recombinase; DNA; DNA repair; DNA replication; Escherichia coli; SDS polyacrylamide gel electrophoresis; X ray crystallography; adenosinetriphosphatase; bacterial proteins; binding sites; enzyme complex; gene targeting; high performance liquid chromatography; mass spectrometry; nucleic acid metabolism; nucleic acid sequence; nucleic acid structure; protein structure function; recombinase

DESCRIPTION (provided by applicant): The overall goal of the proposed research is to use x-ray crystallography and other biochemical tools to understand the structure/function relationships of the E. coil RecA protein, the primary player in homologous recombination (HR). HR is a highly conserved, fundamental biological process that involves Ithe exchange of single-stranded DNA with one strand of a homologous region of duplex DNA. This process is becoming increasingly recognized as an important pathway for the repair of double-stranded DNA breaks, which are frequently generated during DNA replication and by environmental factors. HR is also potentially useful as a tool in the treatment of genetic disorders by gene therapy. RecA, a DNA-dependent ATPase, catalyzes the central strand-exchange step of HR by forming a helical polymer on ssDNA, facilitating a search for a homologous region of duplex DNA, and exchanging strands. E. coil RecA is 30% identical in amino acid sequence to the human enzyme (Rad51) and therefore will have a closely related biochemical mechanism. While the genetics and biochemistry of RecA have been studied for years, there is a major gap in the structural biology of RecA, and many aspects of the biochemical mechanism remain poorly understood. Although there is a x-ray structure of RecA in a helical form, the structure does not include DNA, and therefore does not show how the protein interacts with ssDNA and dsDNA substrates. This proposal outlines several approaches towards crystallizing RecA in complex with its ssDNA substrate. The work is also aimed at crystallizing RecA in the various conformational states, depending on the bound nucleotide, that are relevant to its catalytic mechanism. This work is important not only because the closely related human enzymes are relevant to disease, but also because the biochemical activity of the RecA protein is of fundamental importance to a broad class of enzymes- the helicases- that use a RecA-like structural scaffold to couple the energy of ATP-hydrolysis to DNA unwinding. Thus, understanding the mechanism of action of RecA will contribute greatly to our general knowledge of enzymes involved in virtually all aspects of DNA metabolism.

描述（由申请人提供）： 拟议研究的总体目标是使用X射线晶体学和其他生化工具来了解E. coil RecA蛋白的结构/功能关系，E。Coil RecA蛋白（同源重组（HR）的主要参与者（HR）。人力资源是一种高度保守的基本生物学过程，涉及与单链DNA与双链DNA同源区域的一条链交换。这个过程越来越被公认为是修复双链DNA断裂的重要途径，这是在DNA复制和环境因素中经常产生的。人力资源也可能是通过基因治疗治疗遗传疾病的工具。 RECA是一种DNA依赖性ATPase，通过在ssDNA上形成螺旋聚合物，促进HR的中央链 - 交换步骤，从而促进搜索双链DNA的同源区域并交换链。 E. coil RECA在氨基酸序列中与人酶相同（RAD51），因此将具有密切相关的生化机制。虽然已经研究了RECA的遗传学和生物化学多年，但RECA的结构生物学存在很大的差距，并且生化机制的许多方面仍然了解不足。尽管有螺旋形式的RECA的X射线结构，但该结构不包括DNA，因此没有显示蛋白质如何与ssDNA和DSDNA底物相互作用。该提案概述了与其ssDNA底物复合物结晶RECA的几种方法。这项工作还旨在使各种构象状态的RECA结晶，具体取决于与其催化机理相关的结合核苷酸。 这项工作很重要，不仅是因为与疾病密切相关的人类酶，而且还因为RECA蛋白的生化活性对广泛的酶 - 使用类似RECA的结构性支架来使ATP-Hydrolsy分析的能量促进DNA的能量至关重要。因此，了解RECA的作用机理将极大地有助于我们对参与DNA代谢各个方面的酶的一般知识。