Structural Studies of RecA-DNA Complexes

描述（由申请人提供）： 这项研究的总体目标是利用X射线晶体学和其他生物化学工具来了解E. coil RecA蛋白是同源重组（HR）的主要参与者。HR是一种高度保守的基本生物学过程，涉及单链DNA与双链体DNA同源区的一条链的交换。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也可能用作通过基因疗法治疗遗传性疾病的工具。 RecA是一种DNA依赖性ATP酶，通过在ssDNA上形成螺旋聚合物，促进寻找双链体DNA的同源区域和交换链来催化HR的中心链交换步骤。E. coil RecA在氨基酸序列上与人酶（Rad 51）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已经研究了多年，但RecA的结构生物学存在重大空白，并且生化机制的许多方面仍然知之甚少。虽然RecA的X射线结构是螺旋状的，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ssDNA底物复合物结晶的方法。这项工作的目的也是结晶RecA在各种构象状态，这取决于结合的核苷酸，这是相关的催化机制。 这项工作很重要，不仅因为与疾病密切相关的人类酶，而且因为RecA蛋白的生物化学活性对一大类酶-解旋酶-具有根本的重要性，这些酶使用RecA样结构支架将ATP水解的能量与DNA解旋结合起来。因此，了解RecA的作用机制将极大地有助于我们对参与DNA代谢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.