Structural Studies of RecA-DNA Complexes

描述(由申请人提供)： 这项拟议研究的总体目标是使用X射线结晶学和其他生化工具来了解同源重组(HR)中的主要参与者E.coil RecA蛋白的结构/功能关系。HR是一个高度保守的基本生物学过程，它涉及单链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复制过程中产生，并受到环境因素的影响。HR在通过基因疗法治疗遗传性疾病方面也有潜在的有用工具。 RecA是一种依赖于DNA的ATPase，它通过在单链DNA上形成螺旋聚合物，促进双链DNA同源区域的搜索和链交换来催化HR的中心链交换步骤。大肠杆菌RecA的氨基酸序列与人酶(RAD51)有30%的同源性，因此将具有密切相关的生化机制。虽然RecA的遗传学和生物化学已被研究多年，但在RecA的结构生物学方面仍存在很大差距，对其生化机制的许多方面仍知之甚少。虽然存在螺旋形式的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。该提案概述了几种使RecA与其单链DNA底物形成络合物结晶的方法。这项工作还旨在使RecA在各种构象状态下结晶，这取决于与其催化机制相关的结合核苷酸。 这项工作之所以重要，不仅是因为与人类密切相关的酶与疾病相关，还因为RecA蛋白的生化活性对一大类酶--解旋酶--具有根本重要性，这些酶使用一种类似RecA的结构支架将ATP水解的能量耦合到DNA解离。因此，了解RecA的作用机制将有助于我们对参与DNA代谢的几乎所有方面的酶的一般知识。

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.