Enzymatic Mechanisms of DNA Helicases

描述(申请人提供)：DNA解旋酶催化依赖NIP的双链DNA解离，以提供单链DNA(SsDNA)，用作DNA交易中的模板或反应中间体。我们的工作主要集中在大肠杆菌和发芽酵母中的DNA解旋酶上。这项研究计划的长期目标是从酶和分子方面了解几种重要的DNA解旋酶在大肠杆菌和酵母中的作用机制和细胞作用。在下一个资助期间，我们的重点将是来自大肠杆菌的DNA解旋酶II和IV，以及来自酵母的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，这两个蛋白质在维持基因组稳定方面发挥关键作用。我们将用生化方法确定MutL刺激UvrD催化解离的机制。此外，我们将通过基因研究，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。第三个目标建议使用一种新的生化方法来鉴定与UvrD和解旋酶IV相互作用并调节其活性的蛋白质。我们将在活体中将目标蛋白生物素化，并在细胞提取物的下拉试验中识别相互作用的蛋白。这些蛋白质对每个解旋酶的生化活性的影响将在生化和遗传实验中进行评估。这将进一步阐明这些蛋白质在细胞中扮演的角色。最后两个目标集中在来自萌芽酵母的两个DNA解旋酶上。Sgs1p已经在杆状病毒中以全长蛋白的形式表达，并将在解离机制(过程与分布)、底物偏好(DNA与RNA与DNA-RNA)和底物结构方面被彻底表征为解旋酶。此外，还将用生化方法研究SGS1p与拓扑异构酶I11的相互作用。初步研究表明Topo Ill调控SGS1p的生化活性。这些研究将提供与SGS1p在维持基因组稳定中的作用相关的更多信息。最后，线粒体解旋酶Hmi1p已被鉴定，并在遗传学研究中具有部分特征。目前还缺乏对该蛋白的全面生化描述，并将阐明该蛋白在线粒体DNA新陈代谢中所起的作用。该蛋白将在生化检测中得到表达和充分鉴定。

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.