Dissecting the structural basis for regulation of bacterial DNA polymerase III

描述（由申请人提供）：即使经过几十年的研究，我们对DNA复制中前导链和滞后链合成功能偶联的结构基础所知甚少。一个基本的问题是聚合酶活性位点如何与其他亚基偶联，以在前导链和滞后链合成之间提供适当的校对和协调。我的目标是利用结构和生化研究以及实时可视化相结合的方法，了解细菌聚合酶III系统中这一复杂动态过程的分子基础。本提案的具体目的是：1)确定聚合酶回收过程中工艺转换的结构基础。将确定与聚合酶亚基a结合的tac复合物的结构。2)阐明校对亚基调控pol III活性的机制。初步数据显示，校对亚基与聚合酶活性位点之间可能存在功能连接。本征聚合活性将在存在和不存在校对亚基e的情况下进行测量，以检验校对单元激活聚合的假设。聚合酶杂交体将被制造和表征，以了解活性位点调控原理。最后，为了了解聚合酶和校对之间的相互作用，将开发一种单分子测定方法。3)确定e亚基激活复制校对的结构基础。为了了解校对亚基e影响聚合酶活性的机制，以及pol III从聚合到外核分解转换的结构基础，将解决a-e复合物的结构。公共卫生相关性：了解DNA复制调控的结构基础将具有很大的治疗效用。结合和抑制聚合酶的小分子可能被证明是有用的抗生素。此外，DNA复制缺陷与癌症和神经退行性疾病有关，进一步突出了这一过程对健康科学的重要性。

DESCRIPTION (provided by applicant): Even after decades of study, remarkably little is known about the structural basis for the functional coupling of leading- and lagging-strand synthesis in DNA replication. A fundamental question regards how the polymerase active site couples to other subunits to provide proper proofreading and coordination between leading- and lagging-strand syntheses. My objective is to understand the molecular underpinnings of this complex and dynamic process in the bacterial polymerase III system using a combination of structural and biochemical studies and real-time visualization. The specific aims of this proposal are to: 1) Determine the structural basis for the processivity switch in polymerase recycling. The structure of the complex of tauC bound to the polymerase subunit, a, will be determined. 2) Elucidate the mechanism by which the proofreading subunit modulates pol III activity. Preliminary data shows that there is likely a functional connectivity between the proofreading subunit and polymerase active site. Intrinsic polymerization activity will be measured in the presence and absence of the proofreading subunit, e, to test the hypothesis that the proofreading unit activates polymerization. Polymerase hybrids will be made and characterized to understand active site regulation principles. Finally, a single molecule assay will be developed in order to understand the interplay between the polymerase and proofreading. 3) Determine the structural basis for activation of replication and proofreading by the e subunit. The structure of the a-e complex will be solved in order to understand the mechanism by which the proofreading subunit, e, influences polymerase activity, and the structural basis for the switch from polymerization to exonucleolysis in pol III. PUBLIC HEALTH RELEVANCE: Understanding the structural basis for regulation of DNA replication will have great therapeutic utility. Small molecules that bind and inhibit the polymerase could prove useful as antibiotics. Moreover, defects in DNA replication have been associated with cancer and neurodegenerative disorders, further highlighting the importance of this process to health science.