Enzymatic Mechanisms of DNA Helicases

DNA 解旋酶的酶促机制

• 批准号: 6544326
• 负责人: STEVEN W MATSON
• 金额: $ 24.14万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6544326
• 关键词: DNA repair; DNA replication; DNA topoisomerases; Escherichia coli; Saccharomyces cerevisiae; bacterial genetics; bacterial proteins; enzyme mechanism; fungal genetics; fungal proteins; helicase; molecular cloning; mutant; protein structure function

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.

描述（由申请人提供）：DNA解旋酶催化双链体DNA的NIP水解依赖性解旋，以提供单链DNA（ssDNA），用作DNA转换中的模板或反应中间体。我们的工作主要集中在大肠杆菌中的DNA解旋酶。大肠杆菌和芽殖酵母酿酒酵母。本研究计划的长期目标是从酶和分子方面了解大肠杆菌中几种重要的DNA解旋酶的作用机制和细胞作用。大肠杆菌和酵母菌。在下一个资助期内，我们的重点将是来自E. coli中的Sgs 1 p和Hmi 1 p。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，这两种蛋白质在维持基因组稳定性方面发挥着关键作用。我们将确定MutL刺激UVRD催化解旋使用生化方法的机制。此外，我们将通过遗传学研究，通过鉴定不能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。第三个目标提出使用一种新的生物化学方法鉴定与UvrD和解旋酶IV相互作用并调节其活性的蛋白质。我们将在体内生物素化靶蛋白，并在细胞提取物的下拉测定中鉴定相互作用的蛋白质。这些蛋白质对每种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发挥的作用。最后两个目标集中在两个DNA解旋酶从芽殖酵母。Sgs 1 p已在杆状病毒中表达为全长蛋白，并将在解旋机制（进行性与分布性）、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，Sgs 1 p和拓扑异构酶III之间的相互作用将使用生物化学方法进行研究。初步研究表明，拓扑结构III调制的生化活性的SGS 1 P。这些研究将提供更多的信息相关的SGS 1 P在维持基因组稳定性的作用。最后，线粒体解旋酶，Hmi 1 p已被确定和部分特征的遗传研究。缺乏对这种蛋白质的彻底的生物化学描述，这将有助于阐明这种蛋白质在mtDNA代谢中的作用。该蛋白质将在生物化学测定中表达和充分表征。