Dissecting the structural basis for regulation of bacterial DNA polymerase III

描述(申请人提供)：即使经过几十年的研究，人们对DNA复制中领先和滞后链合成的功能耦合的结构基础仍然知之甚少。一个基本的问题是聚合酶活性位点如何与其他亚单位偶联，以便在领先和滞后的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与实时可视化相结合的方法，了解细菌聚合酶III系统中这一复杂和动态过程的分子基础。这项建议的具体目的是：1)确定聚合酶循环中加工性转换的结构基础。将确定与聚合酶亚单位a结合的tauC复合体的结构。2)阐明校对亚基调控polIII活性的机制。初步数据显示，校对亚基和聚合酶活性位点之间可能存在功能连接。将在存在和不存在校对亚基的情况下测量固有聚合活性，以检验校对单元激活聚合的假设。将制造聚合酶杂交体，并对其进行表征，以了解活性部位调节原理。最后，将开发一种单分子分析，以了解聚合酶和校对之间的相互作用。3)确定e亚基激活复制和校对的结构基础。A-e复合体的结构将被解决，以便了解校对亚基e影响聚合酶活性的机制，以及在PolIII中从聚合转换到核外溶解的结构基础。公共卫生相关性：了解DNA复制调控的结构基础将具有巨大的治疗作用。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.