MECHANISTIC STUDIES OF HEXAMERIC HELICASES

描述：（逐字来自申请人的摘要）解旋酶是马达 利用NTP水解产生的自由能来沿着和 分离双链DNA/RNA链。解旋酶中的缺陷已被证明会导致 遗传不稳定性表现在癌症和早产儿等疾病中， 人类的衰老为了了解解旋酶的酶机制，我们 研究六聚体类解旋酶的两个成员--DNA 来自噬菌体T7（gp 4A '）的解旋酶-引发酶和转录终止子Rho 来自大肠杆菌在上一个资助期内进行的NTP结合/水解研究 和DNA结合表明，六聚体亚基协同作用，并基于 在这些研究中，我们提出了一个3位顺序机制。在该机制中， 三个催化位点以顺序方式水解NTP， DNA结合-释放步骤和解旋酶在ssDNA上的定向移动。 根据单次周转DNA解旋研究，在上一个赠款期， 我们提出了一个“DNA排除”模型。在这个模型中，解旋酶亚基 仅包围叉状DNA的滞后链而排除前导链 从中央通道。我们假设解旋酶转位 在5 '-3'方向上的滞后链上单向地，但是 dsDNA解旋的机制尚不清楚。下一次资助的研究 期间将研究合作NTT催化和DNA解旋 机制等这些研究的具体目标如下。1)以确定 NT的机制？T7 gp 4A和E.大肠杆菌Rho六聚体 结合到DNA/RNA，使用单周转和前稳态NTR动力学。（二） 为了表征所选的T7 gp 4A '的“非偶联”突变体，以深入了解 在将NTR活性偶联到 放松3)使用蛋白质-DNA光交联研究来表征 T7 gp 4A '六聚体与ssDNA和fork-DNA的相互作用，并绘制了T7 gp 4A'六聚体与ssDNA和fork-DNA的相互作用图。 与底物DNA的特定部分相互作用的蛋白质区域。4)到 通过确定步长、步进、 速率，和使用单周转实验解旋的持续性， 首先在不存在T7 DNA聚合酶的情况下，然后在存在T7 DNA聚合酶的情况下。

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.