Molecular Analysis of Tral IE. Coli Helicase 1) Function

描述（由申请人提供）：细菌偶联是一种将偶联质粒从供体转移到受体的过程。环形质粒作为单链DNA转移；因此，一条质粒链必须在供体中断裂，在受体中结扎。对于共轭质粒F， TraI是切割和连接过程的核心参与者。TraI是一种松弛酶或缺口酶，具有显著的序列特异性，可切割单链质粒DNA。当TraI参与一种称为松弛体的多蛋白复合物时，导致TraI和DNA链之间形成稳定链接的DNA缺口就会发生。除了松弛酶活性外，TraI还具有解旋酶活性。有效的转移需要松弛酶和解旋酶活性包含在同一蛋白中，这些活性之间的切换可能是F共轭转移的重要调控步骤。在F转移的一个模型中，TraI将质粒DNA作为松弛体的一部分切割，在接收到启动转移的信号时解离，将DNA的前端从供体引导到受体，利用其解旋酶活性沿着进入的DNA进行跟踪，并将质粒末端连接在一起完成转移。利用遗传、生化、单分子荧光和结构技术的结合，我们提出了旨在回答有关TraI和偶联起始的几个关键问题的实验：我们能否实时观察细胞中的单个松弛体？如果是这样，相对于DNA运输的共轭孔，供体细胞内的松弛体位于哪里？当供体和受体相互作用时，松弛体的位置会改变吗？形成松弛体需要TraI的哪些特征？在转账过程中TraI是否转移到收件人，如果是，我们能否在收件人中检测到TraI ？如果TraI被转移，它是以折叠状态还是变性状态转移的？如果变性，VirB4同系物TraC是否起展开酶的作用？当TraI与它的松弛酶和解旋酶DNA底物相互作用时，它的构象会改变吗？这种构象变化或DNA与TraI的松弛和解旋酶区结合的负协同性是否可以解释TraI在不同角色之间的转换？该研究不仅将提供对共轭转移所需的分子机制的见解，而且还将有助于我们对大型多功能蛋白的调控机制的一般理解。细菌偶联促进了细菌之间的遗传交换，有助于肠道病原体的基因组多样化和进化。结合质粒还可以通过生物膜形成和其他机制参与细菌的发病，是基因治疗过程中促进基因替代的潜在工具。拟议的实验将提供对结合机制的更好理解，最终允许设计用于改善人类健康的操作。

DESCRIPTION (provided by applicant): Bacterial conjugation is a process whereby a conjugative plasmid is transferred from a donor to a recipient. The circular plasmid is transferred as single-stranded DNA; therefore one plasmid strand must be cleaved in the donor and ligated in the recipient. For conjugative plasmid F, TraI is the central player in the cleavage and ligation processes. TraI, a relaxase or nickase, cleaves single-stranded plasmid DNA with remarkable sequence specificity. DNA nicking, which causes formation of a stable linkage between TraI and the DNA strand, occurs while TraI participates in a multi-protein complex called the relaxosome. In addition to its relaxase activity, TraI possesses a helicase activity. Efficient transfer requires that relaxase and helicase activities be contained within the same protein, and a switch between these activities may be an important regulatory step in F conjugative transfer. In one model for F transfer, TraI cleaves plasmid DNA as part of the relaxosome, dissociates upon receiving a signal initiating transfer, pilots the leading end of the DNA out of the donor and into the recipient, tracks along the incoming DNA using its helicase activity, and ligates the plasmid ends together to conclude transfer. Using a combination of genetic, biochemical, single molecule fluorescence and structural techniques, we propose experiments designed to answer several key questions about TraI and conjugation initiation: Can we observe individual relaxosomes in a cell in real time? If so, where is the relaxosome located within the donor cell relative to the conjugative pore through which the DNA is transported? Does the relaxosome location change when donors and recipients interact? What characteristics of TraI are required to form the relaxosome? Is TraI transferred to the recipient during transfer, and if so can we detect TraI in the recipient? If TraI is transferred, is it transferred in a folded or denatured state? If denatured, does the VirB4 homologue TraC act as an unfoldase? Does the conformation of TraI change when it interacts with its relaxase and its helicase DNA substrates? Could such a conformational change or could negative cooperativity of binding of DNA to the relaxase and helicase regions of TraI explain the conversion between roles of TraI? The research will not only provide insight into the molecular mechanisms required for conjugative transfer, but they will also contribute to our general understanding of the regulatory mechanisms of large multifunctional proteins. Bacterial conjugation facilitates genetic exchange between bacteria, assisting genome diversification and evolution of enteric pathogens. Conjugative plasmids also can contribute to bacterial pathogenesis via biofilm formation and other mechanisms, and are potential tools for facilitating gene replacement during gene therapy. The proposed experiments will provide a better understanding of the mechanism of conjugation, eventually allowing manipulations designed to improve human health.