ILLEGITIMATE RECOMBINATION BY DRUG RESISTANCE ELEMENTS

这一建议涉及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 复制和细菌细胞周期和性质的转座 转座子Tn 10及其组分IS 10序列。 I. Dam甲基化在大肠杆菌的起源中起着关键作用， 复制的 复制开始后，半甲基化的 Origin被隔离约15分钟，然后被释放。 这 隔离被认为在防止二次复制中起作用 启动，也可能在染色体分离中发挥作用。 的 dnaA基因的启动子，它在距离原点一分钟的地方定位， 类似地被隔离;这种隔离可能在防止 二次启动和/或作为复制的细胞范围信号的一部分 已经启动。 我们建议调查扣押的性质， oriC和dnaA，从螯合释放的性质，以及 在DNA A的隔离。 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 在dnaA处螯合所需的顺式显性决定簇， 研究改变dam甲基化酶水平对 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。 二. 转座因子在介导DNA 原核生物内部和之间的重排。 我们建议 研究TN 10/IS 10转座反应的性质， 遗传、生物化学和物理方法相结合。 A.体外和体内机制分析。 我们建议将 分子间转座的要求，决定了 链切割和连接之间的功能依赖性，研究 末端转座子碱基对的特殊作用，继续我们的分析 插入特异性，研究转座的机制基础 长度依赖性，并研究加工的缺口供体分子。 B。IS 10转座酶的结构和功能：物理方法。 我们 建议详细描述两种类型的稳定蛋白质/DNA复合物 我们已经确定了它们是转座反应的中间体。 分析将包括通过干扰确定重要的DNA接触 和足迹分析，蛋白质/DNA化学计量的测定， 通过DNA/蛋白质检查蛋白质结构/功能关系 交联和突触复合体拓扑结构的研究 阵 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. IS 10转座酶的结构和功能：遗传学方法。 我们 将继续分析表型和生化三种类型的 已分离的突变体（SOS+Tnsp、ATS和REM），以分离基因内 前两类突变体的回复突变体，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.