ENZYMATIC MECHANISMS OF E COLI DNA HELICASES

描述(改编自研究人员摘要)：DNA解旋酶 催化NTP水解依赖的双链DNA解链以提供 用作模板或反应的单链DNA(SsDNA) DNA交易的中间人。已鉴定出11种解旋酶 在大肠杆菌中；每一个的细胞作用正在被阐明和结构- 功能研究提供了关于心绞痛机制的信息 放松的反应。这项研究计划的长期目标是 从酶和分子的角度理解其作用机制 以及几种大肠杆菌DNA解旋酶的细胞作用。当前的焦点 是关于DNA解旋酶I和II。第一个目的是提出结构功能 解旋酶的研究II.解旋酶中保守的氨基酸残基 基序将被改变，突变蛋白将被提纯和鉴定， 并在遗传分析中对突变等位基因进行评估。这种方法 提供有关解旋酶II的机制和作用的详细信息。 第二和第三个目标集中在蛋白质之间的相互作用。 涉及解旋酶II.对未能 已经设计出了二聚体。对这些突变体的表征将使 二聚作用在解旋酶反应中的重要性评价 机制和细胞角色。投资促进局还将确定和 直接与解旋酶II相互作用的蛋白质的特性 对这种蛋白质在细胞中扮演的角色的额外了解。这个 第四个目标是解决解旋酶II在DNA复制中的作用。至 到目前为止，这一作用尚未确定，建议进行基因实验。 以提供更多细节。第五个目标建议收购 高分辨率的结构信息来补充结构- 机能研究。解旋酶II-单链DNA复合体的晶体结构 将会被确定。第六个目标建议重组 DNA解旋酶I催化的刻痕/解卷反应 细菌接合。催化的划痕和解卷反应 在上一次拨款中分别进行了评估 句号。令人惊讶的是，不可能将这些结合在一起 反应。初步数据表明对宿主蛋白的需求 以触发解锁。该蛋白将被提纯、鉴定和它的 在划痕/解卷反应和细菌结合中的作用 将通过生物化学和遗传学方法加以阐明。

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.