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

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）： 本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大肠杆菌 RecA 蛋白的结构/功能关系，该蛋白是同源重组 (HR) 的主要参与者。 HR 是一个高度保守的基本生物过程，涉及单链 DNA 与双链 DNA 同源区域的一条链的交换。这一过程越来越被认为是修复双链 DNA 断裂的重要途径，双链 DNA 断裂经常在 DNA 复制过程中和环境因素中产生。 HR 还可以作为基因疗法治疗遗传性疾病的潜在工具。 RecA 是一种 DNA 依赖性 ATP 酶，通过在 ssDNA 上形成螺旋聚合物来催化 HR 的中心链交换步骤，促进双链 DNA 的同源区域的搜索和交换链。大肠杆菌 RecA 与人类酶 (Rad51) 的氨基酸序列有 30% 相同，因此具有密切相关的生化机制。虽然 RecA 的遗传学和生物化学研究已多年，但 RecA 的结构生物学仍存在重大空白，生化机制的许多方面仍知之甚少。尽管 RecA 的 X 射线结构呈螺旋形式，但该结构不包含 DNA，因此无法显示该蛋白质如何与 ssDNA 和 dsDNA 底物相互作用。该提案概述了 RecA 与其 ssDNA 底物复合物结晶的几种方法。这项工作还旨在根据与其催化机制相关的结合核苷酸，以各种构象状态结晶 RecA。 这项工作很重要，不仅因为密切相关的人类酶与疾病相关，还因为 RecA 蛋白的生化活性对于一大类酶（解旋酶）至关重要，解旋酶使用类似 RecA 的结构支架来解旋酶。将 ATP 水解的能量与 DNA 解旋耦合。因此，了解 RecA 的作用机制将极大地有助于我们了解涉及 DNA 代谢几乎所有方面的酶的一般知识。