Mechanism of Conjugative DNA Transfer

描述(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，并可发生在 物种，甚至王国。虽然最早是在50多年前被描述的，但我们仍然 对它周围的分子细节只有基本的了解 DNA转移的重要机制。关于连词的详细知识 因此，机制至关重要。这样做的长期目标是 这个项目是在分子水平上理解 接合DNA转移。先前的研究表明，DNA转移始于 接合质粒中的位点和链特异性缺口(NIC)，它是 然后当单链DNA转移到接受者体内时解开。这个实验室有 使用F质粒作为模型，显示了对两个F-编码的要求 Traip和TraYp蛋白，以及一个宿主编码的蛋白，整合宿主 因子(IHF)，在划痕反应中；随后的解卷还没有 在任何系统中重新组合。提出了四个具体目标。第一个目标将是 TraYp和IHF在转氨酶催化的酯交换反应中的作用 反应。初步数据显示IHF可能相互结合 为启动接合提供分子开关的独家位点 处于打开或关闭状态。将使用化学足迹和IHF来探索这一点 结合位点突变体。此外，TraYp+the可能会改变DNA结构 包围NIC，使DNA具有单链DNA(或非B DNA)特性。2号 目的是重建由以下催化剂催化的耦合的划痕-解卷反应 特拉普。初步研究表明，一种以前未被识别的宿主蛋白是 用来“触发”解开被Tralp窃取的DNA。这种蛋白质将是 纯化，使用生化互补分析，并表征 它与Tralp的相互作用以及它在链转移中的作用。F质粒 模型提供了重建这一关键反应的最佳可能性，因为 已确定解旋酶和特定部位的划痕活性，并 最小的松弛小体已经被重组。关于AIM的3个部分将定义 催化残留物(S)参与位点特异性和链特异性的Traip 酯交换反应。初步结果表明， 两种酪氨酸，Y16和Y23。每种酪氨酸的作用将通过以下方式进行评估 构建特异性突变体，并对每个突变体进行体内外评价。 我们还建议在存在的情况下结晶Tralp转酯酶结构域 以及缺乏寡核苷酸底物以深入了解 转酯酶与底物的相互作用。最终目标将集中在 论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。蛋白质将会是 提纯并用于松弛小体重建研究。总而言之， 从这些实验中获得的结果将增进我们对 结合DNA转移的机理，为今后的研究铺平道路 通过实验观察跨细胞膜的转移。

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.