Enzymatic Mechanisms of DNA Helicases

描述（由申请人提供）：DNA解旋酶催化NIP水解依赖的双链DNA解绕，以提供单链DNA (ssDNA)，用作DNA交易中的模板或反应中间体。我们已经集中精力研究大肠杆菌和酿酒酵母的DNA解旋酶。本研究计划的长期目标是从酶和分子的角度了解大肠杆菌和酵母中几种重要DNA解旋酶的作用机制和细胞作用。我们下一个拨款期的重点将是大肠杆菌的DNA解旋酶II和IV，以及酵母的Sgs1p和Hmi1p。第一个和第二个目标将继续我们的努力，以确定解旋酶II （UvrD）和MutL之间的相互作用，这两个蛋白在维持基因组稳定性中起着关键作用。我们将利用生化方法确定MutL刺激uvrd催化解绕的机制。此外，我们将通过遗传学研究，通过识别无法相互作用的突变体来评估这些蛋白质之间相互作用的重要性。第三个目标是使用一种新的生化方法鉴定与UvrD和解旋酶IV相互作用并调节其活性的蛋白质。我们将在体内对目标蛋白进行生物素化，并在细胞提取物的下拉试验中鉴定相互作用的蛋白。这些蛋白对解旋酶生化活性的影响将在生化和遗传实验中进行评价。这将进一步阐明这些蛋白质在细胞中所起的作用。最后两个目标集中在两个DNA解旋酶从萌芽酵母。Sgs1p已在杆状病毒中作为全长蛋白表达，并将在解绕机制（渐进与分布）、底物偏好（DNA、RNA、DNA-RNA）和底物结构方面完全表征为解旋酶。此外，将利用生化方法研究Sgs1p与拓扑异构酶i之间的相互作用。初步研究表明，topo i可以调节Sgs1 p的生化活性。这些研究将为Sgs1 p在维持基因组稳定性中的作用提供更多的相关信息。最后，线粒体解旋酶Hmi1p已被鉴定并在遗传研究中部分表征。缺乏对该蛋白的全面生化描述，并将阐明该蛋白在mtDNA代谢中的作用。该蛋白将在生化分析中表达和充分表征。

DESCRIPTION (provided by applicant): DNA helicases catalyze NIP hydrolysis-dependent unwinding of duplex DNA to provide single-stranded DNA (ssDNA) for use as a template or reaction intermediate in DNA transactions. We have focused our efforts on DNA helicases in E. coli and the budding yeast Saccharomyces cerevisiae. The long-range goal of this research program is to understand, in enzymatic and molecular terms, the mechanism of action and cellular role of several important DNA helicases in E. coli and yeast. Our focus during the next grant period will be on DNA helicases II and IV from E. coli, and Sgs1p and Hmi1p from yeast. The 1st and 2nd aims will continue our efforts to define the interaction between helicase II (UvrD) and MutL, two proteins that play a critical role in maintaining genomic stability. We will determine the mechanism by which MutL stimulates UvrD-catalyzed unwinding using biochemical approaches. In addition, we will evaluate, through genetic studies, the importance of the interaction between these proteins by identifying mutants that fail to interact. The 3rd aim proposes to identify proteins that interact with and modulate the activity of UvrD and helicase IV using a novel biochemical approach. We will biotinylate the target proteins in viva and identify interacting proteins in pull-down assays from cell extracts. The effect of these proteins on the biochemical activities of each helicase will be evaluated in biochemical and genetic experiments. This will shed additional light on the roles these proteins play in the cell. The final two aims focus on two DNA helicases from budding yeast. The Sgs1p has been expressed as a full length protein in baculovirus and will be thoroughly characterized as a helicase with regard to unwinding mechanism (processive vs. distributive), substrate preferences (DNA vs. RNA vs. DNA-RNA) and substrate structure. In addition, the interaction between Sgs1p and Topoisomerase Ill will be investigated using biochemical methods. Preliminary studies suggest that topo Ill modulates the biochemical activity of Sgs1 p. These studies will provide additional information relevant to the role of Sgs1 p in maintaining genomic stability. Finally, a mitochondrial helicase, Hmi1p has been identified and partially characterized in genetic studies. A thorough biochemical description of this protein is lacking and will shed light on the role this protein plays in mtDNA metabolism. This protein will be expressed and fully characterized in biochemical assays.