Structure and Function of Bacterial RecQ Protein

描述(由申请人提供)： 这项建议的目标是确定RecQ DNA解旋酶使用的分子机制。在细菌和真核生物中，RecQ参与了DNA复制、重组和修复，但这个解旋酶家族所扮演的确切角色仍不清楚。然而，它们的重要性是明确的--三个人类RecQ基因中的一个的突变导致基因组不稳定，在Werner‘s、Bloom’s和Rothmund-Thompson综合征中增加癌症的易感性，以及在Werner‘s和Rothmund-Thompson综合征中导致过早衰老。因此，RecQ蛋白代表了一种将基因组稳定性与疾病状态联系起来的范例，以及用于理解DNA复制、重组和修复之间的联系的范例。这使得对RecQ家族机制的精确、分子理解对基础科学和医学科学都很重要。这项建议中描述的实验旨在通过了解RecQ蛋白在体外和体内的分子作用机制来阐明其生物学功能。该方法将重点放在细菌RecQ蛋白上的生化、高分辨率结构和遗传方法上，以描绘支持酶活性的结构-功能关系。这些实验包括：(1)确定RecQ家族中保守结构域(解旋酶、RecQ-Ct和HRDC结构域)的作用；(2)阐明RecQ的四级和三级结构；(3)在细菌细胞中鉴定含有RecQ的蛋白质复合体，并研究相互作用蛋白对RecQ体外活性的影响。一种多学科的方法将允许在RecQ蛋白的细胞角色和它们的分子作用机制之间建立生化联系。

DESCRIPTION (provided by applicant): The goal of this proposal is to determine the molecular mechanisms used by RecQ DNA helicases. RecQ involvement in DNA replication, recombination, and repair has been demonstrated in both bacteria and eukaryotes, but the precise roles played by this family of helicases remain unclear. Their importance, however, is unambiguous -- mutation of one of three human RecQ genes leads to genome instability and an elevated predisposition to cancer in Werner's, Bloom's, and Rothmund-Thompson syndromes as well as to premature aging in Werner's and Rothmund-Thompson syndromes. RecQ proteins, therefore, represent a paradigm linking genome stability to disease states as well as for understanding the connections between DNA replication, recombination, and repair. This makes a precise, molecular understanding of the mechanisms of the RecQ family important to both basic and medical science. The experiments described in this proposal aim to elucidate the biological functions of RecQ proteins by resolving their molecular mechanisms of action in vitro and in viva. The approach focuses biochemical, high-resolution structural, and genetic methods on bacterial RecQ proteins to delineate the structure-function relationship that supports activity in the enzyme. These experiments include: (1) a determination of the roles of the conserved domains within the RecQ family (helicase, RecQ-Ct, and HRDC domains), (2) elucidation of the quaternary and tertiary structures of RecQ, and (3) identification of RecQ-containing protein complexes in bacterial cells and a study of the effects of interacting proteins on RecQ activity in vitro. A multidisciplinary approach will allow biochemical links to be made between the cellular roles of RecQ proteins and their molecular mechanisms of action.