ENZYMATIC MECHANISMS OF E COLI DNA HELICASES

大肠杆菌 DNA 解旋酶的酶促机制

• 批准号: 2701512
• 负责人: STEVEN W MATSON
• 金额: $ 21.9万
• 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 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701512
• 关键词: DNA repair; DNA replication; Escherichia coli; bacterial genetics; bacterial proteins; enzyme mechanism; gene expression; gene mutation; helicase; microorganism conjugation; molecular cloning; mutant; protein structure function; site directed mutagenesis; yeast two hybrid system

DESCRIPTION (adapted from investigator's abstract): DNA helicases catalyze NTP hydrolysis-dependent unwinding of duplex DNA to provide single-stranded DNA (ssDNA) for use as a template or reaction intermediate in DNA transactions. Eleven helicases have been identified in E. coli; the cellular role of each is being elucidated and structure- function studies are providing information regarding the mechanism of the unwinding reaction. 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 E. coli DNA helicases. The current focus is on DNA helicases I and II. The first aim proposes structure-function studies of helicase II. Conserved amino acid residues in helicase motifs will be altered, the mutant protein purified and characterized, and the mutant allele evaluated in genetic assays. This approach provides detailed information on the mechanism and role of helicase II. The second and third aims focus on protein-protein interactions involving helicase II. A genetic screen for mutants that fail to dimerize has been devised. Characterization of these mutants will allow evaluation of the importance of dimerization in helicase reaction mechanisms and cellular roles. The PI will also identify and characterize proteins that interact directly with helicase II to shed additional light on the roles this protein plays in the cell. The fourth aim addresses the role of helicase II in DNA replication. To date, this role is uncharacterized and genetic experiments are proposed to provide additional detail. The fifth aim proposes acquisition of high resolution structural information to complement the structure- function studies. The crystal structure of a helicase II-ssDNA complex will be determined. The sixth aim proposes reconstitution of the nicking/unwinding reaction catalyzed by DNA helicase I to initiate bacterial conjugation. The nicking and unwinding reactions catalyzed by this protein have been evaluated separately during the previous grant period. Surprisingly, it has not been possible to couple these reactions. Preliminary data indicate a requirement for a host protein to trigger unwinding. This protein will be purified, identified and its role in both the nicking/unwinding reaction and bacterial conjugation will be elucidated using biochemical and genetic approaches.

描述（改编自研究者摘要）：DNA解旋酶 催化双链体DNA的NTP水解依赖性解旋， 用作模板或反应单链DNA（ssDNA） DNA交易的中间人。 已经鉴定了11个解旋酶 在大肠大肠杆菌;每个细胞的作用正在阐明和结构- 功能研究提供了关于 解旋反应这项研究计划的长期目标是 从酶和分子的角度来理解 以及几种E. coli DNA解旋酶。 当前焦点 是DNA解旋酶I和II。 第一个目标提出结构-功能 解旋酶的研究Ⅱ. 解旋酶中的保守氨基酸残基 基序将被改变，突变蛋白质将被纯化和表征， 以及在遗传测定中评估的突变等位基因。 这种方法 提供了有关解旋酶II机制和作用的详细信息。 第二和第三个目标集中在蛋白质-蛋白质相互作用 涉及解旋酶II。 一个基因筛选突变体， 已经设计了二聚化。 这些突变体的特征将允许 解旋酶反应中二聚作用重要性的评价 机制和细胞作用。 PI还将识别并 表征直接与解旋酶II相互作用以脱落的蛋白质 进一步阐明这种蛋白质在细胞中的作用。 的 第四个目标是解决解旋酶II在DNA复制中的作用。 到 迄今为止，这种作用尚未得到表征，并提议进行遗传实验 提供更多细节。 第五个目标是收购 高分辨率的结构信息，以补充结构- 功能研究 解旋酶Ⅱ-单链DNA复合物的晶体结构 将被确定。 第六个目标是重建 由DNA解旋酶I催化的切口/解旋反应， 细菌接合 切口和解旋反应催化 在上一次资助中， 期 令人惊讶的是，它一直不可能耦合这些 反应. 初步数据表明需要一种宿主蛋白 引发解旋 该蛋白质将被纯化、鉴定， 在切口/解旋反应和细菌接合中的作用 将使用生物化学和遗传学方法来阐明。