MOLECULAR ANALYSIS OF SITE-SPECIFIC DNA RECOMBINATION

位点特异性DNA重排是一种复杂的反应， 彼此相距很远的核苷酸序列 以高度精确的方式连接在一起。 我们正在研究 沙门氏菌鞭毛相变异系统。 基因 在该系统中，通过特定于站点的 1 kb DNA片段的倒位。 一种无细胞系统， 支持反转反应已经开发。 使用此 系统，所需的蛋白质和序列决定簇 反应的原因已经确定。 最高费率 反转需要三种蛋白质：Hin重组酶， 组蛋白样蛋白HU和一种以前未鉴定的12，000 D 宿主蛋白（因子II）。 DNA底物必须是超螺旋的 并在适当的位置包含两个重组位点， 配置，加上60 bp重组增强子序列。 重组增强子及其相关的 蛋白质（因子II），可以在许多不同的位置发挥作用 相对于重组位点刺激反转 反应超过150倍。 拟议的工作将表征Hin反演系统， 特别关注增强子功能的机制 这系统. 形成的反应中间体和络合物 在反应期间将被分离和表征， 特别是关于联合国的参与或作用， 增强子和因子II蛋白。 因子II的基因将是 进行详细的基因和生化分析。 在 此外，重组缺陷的Hin突变体 将被隔离。 随后对这些突变体的体外分析 蛋白质将使我们能够剖析反应中的各个步骤。 将尝试分离因子II基因中的抑制因子 hin中的特定突变来探测任何可能 发生在因子II和Hin重组酶之间。 最后， F Ⅱ在E.大肠杆菌进行调查， 寻找其他涉及因子II的反应， 将采用增强子样元件。 这些研究应 两者都提供了宝贵的资料，对机制的网站- 特异性DNA重组，增加我们对 涉及增强子和DNA的复杂蛋白质-DNA反应 结构

Site-specific DNA rearrangements are complex reactions in which nucleotide sequences located at great distances from one another are joined together in a highly precise manner. We are studying the flagellar phase variation system of Salmonella. Gene regulation is accomplished in this system by the site-specific inversion of a 1 kb segment of DNA. A cell-free system which supports the inversion reaction has been developed. Using this system, the protein and sequence determinants which are required for the reaction have been identified. Maximum rates of inversion require three proteins: the Hin recombinase, the histone-like protein HU, and a previously unidentified 12,000 D host protein (Factor II). The DNA substrate must be supercoiled and contain two recombination sites in the appropriate configuration, plus a 60 bp recombinational enhancer sequence. The recombinational enhancer, together with its associated protein (Factor II), can function at many different locations relative to the recombination sites to stimulate the inversion reaction over 150-fold. The proposed work will characterize the Hin inversion system, focusing in particular, on the mechanism of enhancer function in this system. Reaction intermediates and complexes formed during the reaction will be isolated and characterized, particularly with regard to the participation or role of the enhancer and Factor II protein. The gene for Factor II will be cloned for a detailed genetic and biochemical analysis. In addition, mutants of Hin which are defective in recombination will be isolated. The subsequent in vitro analysis of these mutant proteins will allow us to dissect the various steps in the reaction. Attempts will be made to isolate suppressors in the Factor II gene of specific mutations in hin to probe any interactions which may be occuring between Factor II and the Hin recombinase. Finally, the role of Factor II in the life of E. coli will be investigated and a search for other reactions involving Factor II acting via enhancer-like elements will be undertaken. These studies should both provide valuable information on the mechanism of site- specific DNA recombination and increase our understanding of complex protein-DNA reactions involving enhancers and DNA structure.