MECHANISM OF INSERTION SEQUENCE TRANSLOCATION

提出了两种不同类型的专业化重组的研究。 转座重组，将转座DNA整合到一个 靶位点，将使用两个对比的转座子进行研究， 模型系统 这些是IS 903，通常由 供体破坏性，非复制性途径，以及使用 复制（共整合）途径。 位点特异性重组 两个特定位点之间通过相互的、保守的 断裂团聚反应，将研究使用的决议 GammaDelta通过转座子编码的TnpR蛋白，解离酶共整合。 提出了以下具体项目。 一、换位思考。 1)IS 903转座酶蛋白的纯化与鉴定 和GammaDelta，以体外转座为最终目标。 2)转座子末端和转座酶（a）的相互作用的分析， 转座子末端反向重复序列的突变分析和（B）通过 转座酶蛋白内DNA结合域的鉴定， 分离显示改变的序列特异性的转座酶突变体。 3)进一步分析了IS 903缺失形成的机制， IS 10。 II.特定地点的澄清。 1)协整分解过程的突变分解，（a）通过 开发用于重组中的各个步骤的测定，和（B）通过 分离和表征在每个步骤中特异性缺陷的突变体。 2)详细分析了（a） 解离酶的N-末端结构域和交叉位点，和（B）解离酶的N-末端结构域和交叉位点， C-末端结构域和解离酶结合DNA片段。 3)分析resDNA的表观解离酶诱导的弯曲。

The study of two different types of specialized recombination is proposed. Transpositional recombination, the integration of transposable DNA into a target site, will be investigated using two contrasting transposons as model systems. These are IS903, which generally transposes by a donor-destructive, non-replicative pathway, and GammaDelta which uses a replicative (cointegrate) pathway. Site specific recombination, the recombination between two specific sites by a reciprocal, conservative breakage reunion reaction, will be studied using the resolution of GammaDelta cointegrates by the transposon encoded TnpR protein, resolvase. The following specific projects are proposed. I Transpositional Recombination. 1) Purification and characterization of the transposase proteins of IS903 and GammaDelta, with transposition in vitro as the ultimate goal. 2) Analysis of the interaction of transposon ends and transposase (a) by mutational analysis of the transposon terminal inverted repeats and (b) by identification of DNA binding domains within the transposase protein by isolation of mutants of transposase that show altered sequence specificity. 3) Further analysis of the mechanism of deletion formation by IS903 and IS10. II Site-specific Recombination. 1) Mutational dissection of the cointegrate resolution process, (a) by developing assays for the various steps in recombination and (b)\by isolating and characterizing mutants specifically defective in each step. 2) Detailed analysis of the molecular interactions between (a) the N-terminal domain of resolvase and the crossover site, and (b) the C-terminal domain and the resolvase-binding DNA segment. 3) Analysis of the apparent resolvase-induced bending of res DNA.