Mechanism of Insertion Sequence Translocation

描述(由申请人提供)：本项目的总体目标是 尽可能详细地了解两种不同的 与转座DNA元件相关的各种专门化重组： 定点重组和转位重组。模型系统 对于特定部位的重组--一种断裂-交换-团聚反应 两个特定的站点-是协整的分解(乘积 伽马三角洲转座子的换位复合) 解析酶，丝氨酸重组酶的原型。转位复合 将使用D，D(35)E超家族的转座子Tn552进行研究。 伽马三角洲分解酶介导的位点特异性重组。 一个主要的目标是阐明突触复合体的结构 (含有两个120碱基的酶切位点和6个二聚体的分解酶) 重组就会发生。我们为此制定了一个新的结构模型 基于结晶学的拆分酶二聚体的配合物和详细的 二聚体间相互作用的知识。各种方法，特别是 荧光共振能量转移(FRET)，将两者用于检测 对这个(和相反的)模型的预测，也是为了探索 伴随蛋白的分解酶亚单位或结构域的重排 催化功能的激活和链交换的过程。一个 另一种相关丝氨酸重组酶转座酶的研究 幽门螺杆菌元件IS60 7也将启动。 Tn552的转位重组。 我们建立了一种有效的Tn552的体外链转移反应， 使用TnpA转座酶和带有预切末端的转座子底物。我们 打算重点研究辅助转座蛋白TnpB在 激活TnpA(允许它切割未切割的转座子末端)和In 换位豁免权。

DESCRIPTION (Provided by applicant): The overall goal of this project is to understand, in as much detail as possible, the mechanisms of two different kinds of specialized recombination associated with transposable DNA elements: site-specific recombination and transpositional recombination. The model system for site-specific recombination - a breakage-exchange-reunion reaction between two specific sites - is the resolution of cointegrates (the product of transpositional recombination by the gamma delta transposon) by the gamma delta resolvase, prototype of the serine recombinases. Transpositional recombination will be studied using a transposon of the D,D(35)E superfamily, Tn552. Site-specific recombination mediated by the gamma delta resolvase. A primary goal is to elucidate the architecture of the synaptic complex (containing two 120 bp ressites and 6 dimers of resolvase) within which recombination occurs. We have formulated a new structural model for this complex based on crystallography of the resolvase dimer and a detailed knowledge of interdimer interactions. A variety of approaches, particularly fluorescence resonance energy transfer (FRET), will be used both to test predictions of this (and an opposing) model, and also to probe for large rearrangements of the resolvase subunits or domains that accompany the activation of catalytic functions and the process of strand exchange. An investigation of another related serine recombinase, the transposase of the Helicobactor pylori element, IS60 7, will also be initiated. Transpositional recombination by Tn552. We have developed an efficient in vitro strand transfer reaction for Tn552, using the TnpA transposase and a transposon substrate with pre-cleaved ends. We intend to focus on the role of the accessory transposition protein, TnpB in activation of TnpA (allowing it to cleave uncleaved transposon ends) and in transposition immunity.