Mechanistic Studies of Hexameric Helicases

说明（由申请人提供）解旋酶是马达蛋白，其利用来自NTP水解的自由能沿沿着核酸移位并分离dsDNA或RNA的链、催化分支迁移或除去与核酸结合的蛋白。解旋酶的这些功能在大多数DNA和RNA代谢过程中是必需的;因此，解旋酶是普遍存在的，并且解旋酶中的某些突变导致遗传不稳定性和疾病，例如人类中许多形式的癌症和过早衰老。在上一个资助期，我们做了全面的努力，以了解三种不同的解旋酶的酶机制：环形T7解旋酶和Rho 蛋白和超家族II丙型肝炎病毒解旋酶。许多拟议的研究将测试从最后一个赠款期产生的假设。动力学分析两个不同的环形解旋酶的目的是实现一个详细的和一般的了解工作的六聚体解旋酶。该六聚体具有六个潜在的NTP酶切位点，可以水解具有不同序列的NTP。我们将NTR反应分解为几个步骤，并确定了限速步骤。在具体目标1中，我们将研究六聚体T7解旋酶和E.通过系统地表征不可水解的核苷酸类似物对NTR的抑制，来研究大肠杆菌Rho在NTP水解中的作用。我们还将研究含有一个或多个功能缺陷突变亚基的T7解旋酶的交联和确定的混合六聚体的活性。ssDNA易位率和dsDNA解旋率的测量表明，T7解旋酶的运动受到双链DNA的限制。在目标2中，我们将表征解旋反应和NTP与链分离的偶联，以确定解旋酶的活性是否与NTP水解紧密偶联，或者解旋酶是否经历例如，在解卷接合处空转或向后运动，从而需要额外的NTP。在目的3中，我们将研究T7解旋酶-T7 DNA聚合酶复合物在复制叉处的协同作用。我们将研究T7 DNA聚合酶激活解旋酶活性的机制，并研究解旋酶-聚合酶复合物对DNA合成的保真度。

DESCRIPTION (provided by applicant) Helicases are motor proteins that use the free energy from NTP hydrolysis to translocate along nucleic acids and to separate the strands of dsDNA or RNA, catalyze branch migration, or remove proteins bound to nucleic acids. These functions of helicases are required in most DNA and RNA metabolic processes; therefore, helicases are ubiquitous and certain mutations in helicases lead to genetic instabilities and diseases such as many forms of cancer and premature aging in humans. In the previous grant period, we made a comprehensive effort to understand the enzymatic mechanisms of three different helicases: ring-shaped T7 helicase and Rho proteins, and superfamily II hepatitis C virus helicase. Many of the proposed studies will test the hypotheses generated from the last grant period. Kinetic analysis of two different ring-shaped helicases aims to achieve both a detailed and a general understanding of the workings of hexameric helicases. The hexamer has six potential NTPase sites that can hydrolyze NTP with various sequences. We have dissected the NTPase reaction into several steps and determined the rate limiting step. In specific Aim 1, we will investigate cooperativity amongst the subunits of hexameric T7 helicase and E. coli Rho in NTP hydrolysis by systematically characterizing the inhibition of NTPase by the non-hydrolyzable nucleotide analogs. We will also study the activities of crosslinked and defined mixed hexamers of T7 helicase containing one or more functionally defective mutant subunit. Measurement of the ssDNA translocation rate and the dsDNA unwinding rate has shown that the movement of T7 helicase is restricted by duplex DNA. In Aim 2, we will characterize the unwinding reaction and the coupling of NTPase to strand separation to determine if the helicase's activity is tightly coupled to NTP hydrolysis or if the helicase undergoes e.g., idling or backward movement at the unwinding junction, thereby requiring additional NTP. In Aim 3, we will study the cooperative actions of the T7 helicase-T7 DNA polymerase complex at the replication fork. We will investigate the mechanism by which T7 DNA polymerase activates the helicase activity and investigate the fidelity of DNA synthesis by the helicase-polymerase complex.