MECHANISM OF INSERTION SEQUENCE TRANSLOCATION

这个项目的总体目标是尽可能多地了解 尽可能详细地介绍了两种不同类型的专门机制， 重组：转座重组和位点特异性重组 重组 转座重组-整合的一个 将转座DNA元件插入靶位点-将使用 两个对比的转座子作为模型系统。 这是IS 903， 似乎主要通过供体破坏性非复制性 切除-插入途径，和γ δ，它使用一个完全复制的 供体完整存活的途径。 该模型系统用于现场- 特异性重组-两个特定位点之间的重组， 可逆保守断裂-团聚反应-是解决 gamma delta协整函数（gamma delta换位的乘积） 重组）由γ δ编码的解离酶介导。 一、换位思考 对于IS 903和γ δ，我们将尝试建立体外系统， 换位，建立在我们对生产的知识增加的基础上 和转座酶蛋白的行为。 灵敏的检测方法，能够检测 链转移中间体以及 转座，将被使用。 将尝试使用基因和 生物化学方法，以确定每个转座酶的区域 负责催化功能和识别异常 转座子末端的长DNA结合位点。 两个转座子特异性 特性也将被调查：强顺式作用 IS 903转座酶的选择性与转座免疫 由伽马德尔塔展示。 II.由γ δ Resolvase介导的位点特异性纯化 主要目的是了解这两者的分子性质 由解离酶与res相互作用形成的核蛋白复合物： 解离体，由3个（可能）解离酶二聚体结合的单个颗粒，和 突触体是一个复合体，有两个成对的静止位点， 交换发生。 催化结构域的晶体结构 resolvase为以下目标提供了结构基础：（a）到 确定负责解离酶单体的二聚体排列， 在每个交叉点处的链切割;（B）以定义各种 解离酶原聚体之间的成对相互作用导致组装 分离体和突触体，决定哪个分离体结合 子网站相互作用，并确定表面的 （c）确定res DNA相对于 催化域的堆积。 另一个目的是了解更多 充分利用DNA识别和结合B的过程， 分解酶

The overall goal of this project is to understand, in as much molecular detail as possible, the mechanisms of two different types of specialized recombination: transpositional recombination and site-specific recombination. Transpositional recombination - the integration of a transposable DNA element into a target site - will be investigated using two contrasting transposons as model systems. These are IS903, which appears to transpose predominantly by a donor-destructive non-replicative excision-insertion pathway, and gamma delta, which uses a fully replicative pathway in which the donor survives intact. The model system for site- specific recombination - the recombination between two specific sites by a reciprocal conservative breakage-reunion reaction - is the resolution of gamma delta cointegrates (the product of gamma delta transpositional recombination) mediated by the gamma delta-encoded resolvase. I.Transpositional Recombination For IS903 and gamma delta we shall attempt to set up in vitro systems for transposition, building on our increased knowledge regarding the production and behavior of the transposase proteins. Sensitive assays, able to detect the strand transfer intermediates as well as the final products of transposition, will be used. Attempts will be made, using genetic and biochemical approaches, to identify the regions of each transposase responsible for catalytic functions and for recognition of the unusually long DNA binding sites at the transposon termini. Two transposon-specific characteristics will also be investigated: the strong cis-acting preference of the IS903 transposase and the transpositional immunity exhibited by gammadelta. II.Site-specific Recombination Mediated by the gamma delta Resolvase The primary aim is to understand the molecular nature of the two nucleoprotein complexes formed by the interaction of resolvase with res: the resolvosome, a single res bound by 3 (presumably) resolvase dimers, and the synaptosome, the complex with two paired res sites within which strand exchange occurs. The crystal structure of the catalytic domain of resolvase provides a structural basis for the objectives which are: (a to identify the dimeric arrangement of resolvase monomers responsible for the strand cleavage at each crossover point; (b) to define the various pairwise interactions between resolvase protomers that result in assembly of the resolvosome and synaptosome, determining which resolvase-bound subsites interact with one another and identifying the surfaces of interaction used; (c) to define the path of the res DNA relative to the packing of the catalytic domains. An additional aim is to understand more fully the process of DNA recognition and binding b the C-terminal domain of resolvase.