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，将可转座的DNA整合到 靶点，将使用两个对比的转座子 模型系统。这些是IS903，它通常由一个 捐赠者破坏性的非复制途径，以及Gammadelta使用 复制(共整合)途径。特定部位的重组， 两个特定位点之间通过一种相互的、保守的 断裂团聚反应，将使用分辨率来研究 Gammadelta通过转座子编码的TnpR蛋白、解析酶进行共整合。 提出了以下具体项目。 I转位重组。 1)IS903转座酶蛋白的纯化与鉴定 和Gammadelta，以体外转座为最终目标。 2)分析转座子末端与转座酶(A)的相互作用 转座子末端反向重复序列的突变分析和(B) 转座酶蛋白中DNA结合区的鉴定 分离显示序列特异性改变的转座酶突变体。 3)进一步分析了IS903和IS903的缺失形成机制 是10。 II位点特异性重组。 1)协整分解过程的突变解剖，(A)通过 为重组中的各个步骤开发分析方法和(B) 分离和鉴定在每一步中特别有缺陷的突变体。 2)详细分析(A)之间的分子相互作用 解析酶的N-末端结构域和交叉位点，以及(B) C末端结构域和解旋酶结合DNA片段。 3)表观裂解酶诱导的RES DNA弯曲的分析。

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.