MECHANISM OF INSERTION SEQUENCE TRANSLOCATION

这个项目的总体目标是理解，在尽可能多的分子 尽可能详细地介绍两种不同类型的专门化机制 重组：转位重组和位点特异性 重组。转位重组--一种 可转座的DNA元素进入目标位置-将使用 两个截然不同的转座子作为模型系统。这些是IS903，它 似乎主要是由供体破坏性的非复制体转置 切除-插入途径，以及使用完全复制的伽马增量 捐献者完好无损地存活的途径。场址模型系统-- 特定重组--两个特定位点之间的重组 互惠保守的破裂--重聚反应--是 伽马三角洲协整(伽马三角洲转位的乘积 重组)，由伽马增量编码的解旋酶介导。 一、转位重组 对于IS903和伽马三角洲，我们将尝试建立体外系统 换位，建立在我们对生产的更多了解的基础上 以及转座酶蛋白的行为。灵敏的分析，能够检测到 链转移中间体以及 换位，将被使用。将进行尝试，使用基因和 生化方法，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。两个转座子专一性 还将调查其特点：强烈的顺应性 IS903转座酶的选择性与转座免疫 由Gammadelta展出。 II.伽马三角洲溶血酶介导的位点特异性重组 主要目的是了解两者的分子性质。 解旋酶与RES相互作用形成的核蛋白复合体： 解旋体是由3个(可能)解离酶二聚体结合的单个Res，以及 突触体，具有两个成对的RES位点的复合体，其中 交换发生了。其催化域的晶体结构 解决方案为以下目标提供了结构基础：(a至 确定起作用的分解酶单体的二聚体排列 在每个交叉点进行链切割；(B)定义不同的 导致组装的分解酶原体间的成对相互作用 解体和突触体，决定与哪个解体结合 子站点之间相互作用，并识别 所使用的相互作用；(C)定义res DNA相对于 催化域的填充。另一个目标是了解更多 完整的DNA识别和结合B-末端结构域的过程 解析器。

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.