MECHANISTIC STUDIES OF HEXAMERIC HELICASES

描述：(逐字摘自申请者的摘要)解旋酶是马达 蛋白质利用NTP水解的自由能沿和 分离dsDNA/RNA链。解旋酶的缺陷已被证明是导致 表现为癌症和早产儿等疾病的遗传不稳定 人类的衰老。为了了解解旋酶的酶机制，我们正在 研究六聚体解旋酶的两个成员--DNA 噬菌体T7解旋酶启动子(gp4A‘)及其转录终止子Rho 来自E.Coli。NTP结合/水解的前一授权期研究 DNA结合显示六聚体亚基协同作用，并基于 在这些研究的基础上，我们提出了三点序贯机制。在这个机制中， 三个催化位依次水解NTP，促进顺序 DNA结合-释放步骤和单链DNA上解旋酶的定向运动。 根据单一营业额DNA平仓研究，在之前的赠款期间， 我们提出了一个“DNA排除”模式。在这个模型中，解旋酶亚基 只包围叉状DNA的滞后链，不包括前导链 从中央频道。我们推测解旋酶易位 在5‘-3’方向的滞后链上单向，但 DsDNA解离的机制尚不清楚。下一笔拨款中的研究 期间将研究合作的NTPase催化和DNA解离 机械装置。这些研究的具体目的如下。1)确定 NT？T7 gp4A和E.ColiRho六聚体催化的水解酶 结合DNA/RNA，使用单一周转和稳态前的NTPase动力学。2) 鉴定精选的T7 gp4A‘非偶联突变体以深入了解 将NTPase活性偶联到 放松。3)利用蛋白质-DNA光交联研究来表征 T7 gp4A‘六聚体与单链DNA和叉链DNA的相互作用 与底物DNA的特定部分相互作用的蛋白质区域。4)至 通过确定步长、步长来表征展开反应 速率，以及单周转实验的退卷工艺性， 首先在没有T7DNA聚合酶的情况下，然后在T7DNA聚合酶存在的情况下。

DESCRIPTION: (Verbatim from the Applicant's Abstract) Helicases are motor proteins that use the free energy from NTP hydrolysis to translocate along and separate the strands of dsDNA/RNA. Defects in helicases have been shown to lead to genetic instability that manifests in diseases such as cancer and premature aging in humans. To understand the enzymatic mechanisms of helicases, we are studying two members of the hexameric class of helicases-the DNA helicase-primase from phage T7 (gp4A') and the transcription terminator Rho from E. coli. Studies in the previous grant period of NTP binding/ hydrolysis and DNA binding showed that the hexamer subunits act cooperatively, and based on these studies we proposed a 3-site sequential mechanism. In this mechanism, three catalytic sites hydrolyze NTP in a sequential manner promoting sequential DNA bind-release steps and directional movement of the helicase on the ssDNA. Based on single turnover DNA unwinding studies, in the previous grant period, we have proposed a "DNA-exclusion" model. In this model, the helicase subunits surround only the lagging strand of the fork-DNA and exclude the leading strand from the central channel. We postulate that the helicase translocates unidirectionally on the lagging strand in the 5'-3' direction, but the mechanism of dsDNA unwinding is yet unknown. The studies in the next grant period will investigate the cooperative NTPase catalysis and the DNA unwinding mechanisms. The specific aims of these studies are as follows. 1) To determine the mechanism of NT? hydrolysis catalyzed by T7 gp4A and E. coli Rho hexamers bound to DNA/RNA, using single-turnover and presteady-state NTPase kinetics. 2) To characterize selected "uncoupled" mutants of T7 gp4A' to gain insights into the enzymatic steps that are critical in coupling the NTPase activity to unwinding. 3) To use protein-DNA photo cross-linking studies to characterize the interactions of T7 gp4A' hexamer with ssDNA and fork-DNA, and map the protein regions that interact with specific parts of the substrate DNAs. 4) To characterize the unwinding reaction by determining the step-size, stepping rate, and the processivity of unwinding using single-turnover experiments, first in the absence and then in the presence of T7 DNA polymerase.