MOLECULAR ANALYSIS OF TRAL (E. COLI HELICASE I) FUNCTION

描述：(申请人提供)：结合是一种重要手段 细菌物种之间的遗传转移和已被牵连的 编码抗生素耐药性的基因传播。这项研究的目的是 程序是为了了解质粒划痕的机制和调控。 细菌接合过程中DNA转移的启动。质粒刻痕，a 转移单质粒DNA链的前提条件是 松弛酶，结合和裂解单链DNA的核酸酶。Trai，The 接合质粒F因子的松弛酶是F接合必不可少的 调职。TRAI同时具有松弛酶活性和解旋酶活性。 后者解开并分离出作为切断链的质粒DNA链 转给收件人。Tral如何能够对Double-Stand采取行动 DNA在体内，以及Trai如何在其松弛酶和解旋酶活性之间转换 都是未知的。我们建议通过实验来回答有关TRAL的四个关键问题 和接合启动：序列切割和转移启动是如何进行的 受ORIT DNA序列和结合它们的蛋白质的影响？什么 蛋白质-蛋白质相互作用影响训练和DNA的起始 转移，以及扰乱它们的影响是什么？蛋白质是如何 识别具有精致序列特异性的单链DNA？何以 TRAI结构和领域组织影响其活动吗？这些问题 将通过体内研究确定Tral蛋白来回答， 影响转移的蛋白质相互作用和质粒DNA序列，以及 将决定这些蛋白质和DNA序列对质粒的影响 划痕、转移启动和转移终止。体外研究将 专注于确定Trai可以识别的DNA序列和扭曲， 构象或功能的Trai-DNA相互作用的能量学 Trai与DNA结合后发生的事件以及结构的作用 以及域组织在确定Trai功能中的作用。

DESCRIPTION: (provided by applicant): Conjugation is an important means of genetic transfer between bacterial species and has been implicated in the spread of genes encoding antibiotic resistance. The goal of this research program is to understand the mechanism and regulation of plasmid nicking and of initiation of DNA transfer in bacterial conjugation. Plasmid nicking, a prerequisite of transfer of a single plasmid DNA strand, is performed by relaxases, nucleases that bind and cleave single-stranded DNA. TraI, the relaxase of conjugative plasmid F Factor, is essential to F conjugative transfer. TraI possesses both a relaxase activity and a helicase activity, the latter unwinding and separating the plasmid DNA strands as the cut strand is transferred to the recipient. How Tral is able to act against double-stranded DNA in vivo, and how TraI converts between its relaxase and helicase activities are unknown. Experiments are proposed to answer four key questions about Tral and conjugation initiation: How is TraI nicking and transfer initiation influenced by oriT DNA sequences and the proteins that bind them? What protein-protein interactions influence TraI nicking and the initiation of DNA transfer, and what is the effect of disrupting them? How does a protein recognize single-stranded DNA with exquisite sequence specificity? How does TraI structure and domain organization influence its activity? These questions will be answered through in vivo studies that will identify Tral proteins, protein interactions and plasmid DNA sequences that influence transfer, and will determine the effects of these proteins and DNA sequences on plasmid nicking, transfer initiation, and transfer termination. In vitro studies will focus on determining the DNA sequences and distortions that TraI can recognize, the energetics of the TraI-DNA interaction, the conformational or functional events that occur subsequent to TraI binding to DNA, and the role of structure and domain organization in determining TraI function.