Mechanism of Conjugative DNA Transfer

描述（由申请人提供）：遗传性状的接合转移是 由广泛的质粒和转座子介导，并且可以发生在 物种甚至王国。虽然在50多年前首次被描述，但我们仍然 我对围绕这一点的分子细节只有初步的了解 DNA转移的重要机制。关于共轭的详细知识 因此，机制至关重要。长期目标是 项目是在分子水平上了解 接合DNA转移。以前的研究表明，DNA转移开始于 接合质粒中的位点和链特异性切口（nic）， 然后随着ssDNA转移到受体中而解开。本实验室曾 显示，使用F质粒作为模型，需要两个F编码的 蛋白，Traip和TraYp，以及一种宿主编码的蛋白，整合宿主 因子（IHF），在切口反应中;随后的解旋尚未被发现。 在任何系统中都是如此。提出了四个具体目标。第一个目标是 重点研究了TraYp和IHF在Tralp催化酯交换反应中的作用 反应初步数据表明，IHF可能与两种相互作用的蛋白质之一结合。 提供用于引发缀合的分子开关的排他性位点， 不是开就是关将使用化学足迹法和IHF对此进行探讨 结合位点突变体。此外，TraYp + tHE可能会改变DNA结构， 包围nic，使DNA具有ssDNA（或非B DNA）特征。第2 目的是重建由以下物质催化的偶联切口-解旋反应： Tralp最初的研究表明，一种以前未被识别的宿主蛋白质， 需要“触发”由Tralp切割的DNA解旋。这种蛋白质将 纯化，使用生化互补测定，并在术语 它与Tralp的相互作用及其在链转移中的作用。F质粒 模型提供了重建这一关键反应的最佳可能性，因为 已经鉴定了解旋酶和位点特异性切口活性， 最小松弛体已重建。3关于目标将定义 Traip中的催化残基参与位点特异性和链特异性 酯交换反应初步结果显示， 两个酪氨酸Y16和Y23每种酪氨酸的作用将通过 构建特异性突变体并在体外和体内评价每种突变体。 我们还建议在存在下结晶Tralp转酯酶结构域 和缺乏寡核苷酸底物，以深入了解 转酯酶与其底物的相互作用。最终目标将集中在 关于TraMp在引发反应中的作用。遗传学研究表明， 这种蛋白质的作用，但缺乏生化细节。蛋白质将 纯化并用于松弛体重建研究。总的来说， 从这些实验中获得的结果将促进我们对 研究了DNA接合转移的机理，为今后的研究奠定了基础 研究跨细胞膜转移的实验。

DESCRIPTION (Provided by applicant): Conjugative transfer of genetic traits is mediated by a wide range of plasmids and transposons, and can occur between species and even kingdoms. Although first described over 50 years ago, we still have only a rudimentary knowledge of the molecular details surrounding this important mechanism for DNA transfer. Detailed knowledge of the conjugative mechanism is, therefore, of critical importance. The long range goal of this project is to understand, at a molecular level, the mechanistic details of conjugative DNA transfer. Previous studies indicate that DNA transfer begins at a site- and strand-specific nick (nic) in the conjugative plasmid, which is then unwound as ssDNA is transferred into the recipient. This laboratory has shown, using the F plasmid as a model, a requirement for two F-encoded proteins, Traip and TraYp, and one host-encoded protein, integration host factor (IHF), in the nicking reaction; subsequent unwinding has not yet been reconstituted in any system. Four specific aims are proposed. The 1st aim will focus on the role of TraYp and IHF in the Tralp-catalyzed transesterification reaction. Preliminary data suggest IHF may bind to one of two mutually exclusive sites that provide a molecular switch for initiating conjugation that is either on or off. This will be explored using chemical footprinting and IHF binding site mutants. In addition, TraYp + tHE may alter the DNA structure surrounding nic such that the DNA has ssDNA (or non-B DNA) character. The 2nd aim is to reconstitute the coupled nicking-unwinding reaction catalyzed by Tralp. Initial studies indicate a previously unrecognized host protein is required to "trigger" unwinding of DNA nicked by Tralp. This protein will be purified, using a biochemical complementation assay, and characterized in terms of its interaction with Tralp and its role in strand transfer. The F plasmid model provides the best possibility of reconstituting this key reaction because the helicase and site-specific nicking activities have been identified and a minimal relaxosome has been reconstituted. The 3 about aim will define the catalytic residue(s) in Traip involved in the site- and strand-specific transesterification reaction. Preliminary results indicate the involvement of two tyrosines, Y16 and Y23. The role of each tyrosine will be evaluated by constructing specific mutants and evaluating each mutant in vitro and in vivo. We also propose to crystallize the Tralp transesterase domain in the presence and absence of an oligonucleotide substrate to gain insight into the interaction of the transesterase with its substrate. The final aim will focus on the role of TraMp in the initiation reaction. Genetic studies indicate a role for this protein but biochemical details are lacking. The protein will be purified and used in relaxosome reconstitution studies. Taken together, the results gained from these experiments will advance our understanding of the mechanism of conjugative DNA transfer and will pave the way for future experiments to look at transfer across the cell membrane.