ENZYMATIC MECHANISMS OF THE E COLI HELICASES

DNA的双螺旋结构要求存在一个 解螺旋的机制，以暴露单链DNA， 用DNA聚合酶作为模板。 所提供的一种机制是 解旋酶：一类酶，即解旋酶，利用化学物质使DNA解旋 NTP水解提供的能量。 五个解旋酶已经被 描述于E.大肠杆菌、解旋酶I、II、III、Rep蛋白和DnaB 蛋白质;每一个在DNA代谢中的确切作用-才刚刚开始 有待阐明。 这项研究计划的长期目标是 从酶和分子的角度来理解 每个解旋酶的作用，并定义其各自的作用， DNA复制、修复、重组和接合。 旋酶 可能参与DNA代谢的各个方面 这使得E.杆菌 解旋酶活性 深入了解特定解旋酶的作用 通过确定DNA底物的需求量， 反应机理的确定和蛋白质的阐明- 蛋白质相互作用 遗传分析补充了这一生化 approach. 解旋酶II和Rep对DNA底物需求的研究 蛋白质已被采用。 额外蛋白质的作用 如大肠coli SSB对解旋反应的影响。 此外，对以前未知的蛋白质的系统搜索 刺激每个解旋酶反应。 对这些蛋白质及其编码基因的鉴定， 提供额外的生物化学和遗传学工具， 每个解旋酶的精确角色。 一种新的解旋酶，75-kDa 解旋酶已经被发现。 生化分析 该酶催化的解旋反应和基因的克隆 将完成编码该解旋酶。 结构功能 利用定点突变对uvrD基因进行的研究， 提出了 ATP酶、解旋酶和DNA结合的鉴定 网站是短期目标。 总之，预计这些 研究将提高我们对复杂反应方案的理解 例如切除修复、重组和DNA复制。 了解每种解旋酶的DNA底物要求， 解旋酶和其他蛋白质之间的蛋白质-蛋白质相互作用的性质 蛋白质，解旋酶之间的关系的研究是 需要了解每个解旋酶在DNA代谢中的作用。

The double helical structure of DNA mandates the existence of a mechanism for unwinding the helix to expose single-stranded DNA for use as a template by DNA polymerase. One mechanism provided is a class of enzymes, the helicases, that unwind DNA using chemical energy provided by NTP hydrolysis. Five helicases have been described in E. coli, helicases I, II, III, Rep protein and DnaB protein; the exact role of each in DNA metabolism-is just beginning to be elucidated. The long-range goal of this research program is to understand, in enzymatic and molecular terms, the mechanism of action of each helicase, and to define their respective roles in DNA replication, repair, recombination and conjugation. Helicases are likely to be involved in all aspects of DNA metabolism necessitating the presence of several enzymes in E. coli with helicase activity. Insight into the role of a specific helicase has been gained by determination of DNA substrate requirements, determination of reaction mechanism and elucidation of protein- protein interactions. Genetic analyses complement this biochemical approach. Studies of DNA substrate requirements for helicases II and Rep protein have been undertaken. The effect of additional proteins such as E. coli SSB on the unwinding reaction will be evaluated. In addition, a systematic search for previously unknown proteins that stimulate each helicase reaction will be carried out. Identification of such proteins, and the genes encoding them, will provide additional biochemical and genetic tools for determining precise roles for each helicase. A new helicase, the 75-kDa helicase has been discovered. Biochemical analysis of the unwinding reaction catalyzed by this enzyme and cloning of the gene encoding this helicase will be accomplished. Structure-function studies of the uvrD gene, using site-directed mutagenesis, are proposed. Identification of the ATPase, helicase and DNA binding sites are short-range goals. In summary, it is expected that these studies will enhance our understanding of complex reaction schemes such as excision repair, recombination and DNA replication. Knowledge of DNA substrate requirements for each helicase, the nature of protein-protein interactions between helicases and other proteins, and study of the relationship between helicases are required to understand the role of each helicase in DNA metabolism.