ILLEGITIMATE RECOMBINATION BY DRUG RESISTANCE ELEMENTS

这项建议解决了染色体行为的两个不同方面 细菌：DNA腺嘌呤(DAM)甲基化在调节DNA中的作用 细菌的复制和细胞周期以及转座的性质 转座子Tn10及其组成为10个序列。 I.Dam甲基化在大肠杆菌的起源中起着关键作用 复制。复制开始后，半甲基化的 Origin被隔离约15分钟，然后释放。这 隔离被认为在防止辅助复制方面发挥了作用 并可能在染色体分离中发挥作用。这个 DNAA基因的启动子距离起点只有一分钟的距离， 类似的隔离；这种隔离可能在防止 二次启动和/或作为细胞范围信号的一部分 已经发起了。我们建议在以下地点调查自动减支的性质 ORIC和DNAA，解除自动减支的性质和作用 DNAA的自动减支计划。为此，我们建议分离出三种类型 影响这一过程的细菌突变，以确定 DNAA的封存所需的顺式主导决定因素，至 研究不同水平的DAM甲基酶对小鼠肝细胞生长的影响。 隔离和辅助复制启动，并调查 将DNAA基因从ORIC移开的生物学后果。 转座元件在DNA调节中起主要作用 原核生物内部和之间的重排。我们建议 研究Tn10/IS10转座反应的性质 基因、生物化学和物理方法的结合。 A.体外和体内的力学分析。我们建议定义 分子间转座的要求，确定和的性质 链切割和连接之间的功能依赖，调查 末端转座子的特殊作用，继续我们的分析 插入特异性，研究转位的机制基础 长度依赖关系，并研究有缝隙的供体分子的加工。 B.IS10转座酶的结构和功能：物理方法。我们 建议详细表征两种稳定的蛋白质/DNA复合体 我们已经确定它们是转座反应的中间产物。 分析将包括通过干扰定义重要的DNA接触 足迹分析，蛋白质/DNA化学计量学的测定， 利用DNA/蛋白质研究蛋白质结构/功能关系 交联化和突触复合体的拓扑学研究 队形。我们还建议寻找不太稳定的转座酶/DNA 可能存在但尚未确定的复合体。 C.IS10转座酶的结构和功能：遗传学方法。我们 将继续分析三种类型的表型和生化 已分离的突变体(SOS+TnSP、ATS和REM)，以分离基因内 前两种类型的突变体，并寻找新的类型 转座酶突变体。

This proposal addresses two different aspects of chromosome behavior in bacteria: the role of DNA adenine (dam) methylation in regulating DNA replication and the bacterial cell cycle and the nature of transposition by transposon Tn10 and its component IS10 sequences. I. Dam methylation plays a critical role at the E.coli origin of replication. Immediately after replication initiation, the hemimethylated origin is sequestered for about 15 minutes and then released. This sequestration is thought to play a role in preventing secondary replication initiations and may also play a role in chromosome segregation. The promoter for the dnaA gene, which maps a minute away from the origin, is similarly sequestered; this sequestration may play a role in preventing secondary initiations and/or be part of a cell-wide signal that replication has initiated. We propose to investigate the nature of sequestration at oriC and dnaA, the nature of the release from sequestration, and the role of sequestration at dnaA. To these ends, we propose to isolate three types of bacterial mutations which affect this process, to determine the cis-dominant determinants required for sequestration at dnaA, to investigate the effects of varying the level of dam methylase on sequestration and secondary replication initiations, and to investigate the biological consequence of moving the dnaA gene away from oriC. II. Transposable elements play a major role in mediating DNA rearrangements within and between prokaryotic organisms. We propose to investigate the nature of the TN10/IS10 transposition reaction through a combination of genetic, biochemical and physical approaches. A. Mechanistic analysis in vitro and in vivo. We propose to define the requirements for intermolecular transposition, determine the nature of and functional dependence among strand cleavages and ligations, investigate the special role of terminal transposon basepairs, continue our analysis of insertion specificity, investigate the mechanistic basis of transposition length dependence, and investigate processing of gapped donor molecules. B. Structure and function of IS10 transposase: physical approaches. We propose to characterize in detail two types of stable protein/DNA complexes that we have identified as intermediates in the transposition reaction. Analysis will include definition of important DNA contacts by interference and footprinting assays, determination of protein/DNA stoichiometry, examination of protein structure/function relationships by DNA/protein crosslinking, and investigation of the topology of synaptic complex formation. We also propose to search for less stable transposase/DNA complexes that are likely to exist but have not yet been identified. C. Structure and function of IS10 transposase: genetic approaches. We will continue to analyze phenotypically and biochemically three types of mutants already isolated (SOS+Tnsp, ATS, and REM), to isolate intragenic revertants of mutants of the first two types, and to search for new types of transposase mutants.