RECA PROTEIN STRUCTURE AND FUNCTION

本研究提案的目标是阐明和表征 对RecA活动很重要的基本相互作用 大肠杆菌的蛋白质。 这种蛋白质是同源性所必需的 依赖性重组，用于调节一组参与 DNA损伤应激反应（SOS反应）和直接参与 导致突变的生化过程。 基本的相互作用 将被研究的是那些涉及i）RecA蛋白亚基 识别，ii）ATP结合和水解，以及iii）识别和 与其他蛋白质如类胡萝卜素阻遏物、细胞莱克萨结合 阻遏物和umuD蛋白，这是一个主要的途径所需要的， 化学诱变 在RecA蛋白的前90个残基内的序列为 将使用亲和层析研究亚基结合， RecA和β-内酰胺酶之间的融合蛋白。 合成的结合 将研究对应于感兴趣区域的肽。 ATP中的两个取代突变（Tyr 264-Phe 264和Tyr 264-Ser 264） 结合位点已经通过定点诱变构建， 在ATP水解、DNA结合和 进行DNA配对（退火、链转移）的能力。 二站点 将分离和表征抑制突变，以便 识别ATP结合域的其他重要元件。 此外，本发明还提供了一种方法， 定点诱变将用于改变具有保守的 TecA蛋白中的一个序列，在许多核苷酸结合中发现 proteins. 蛋白质的性质将在体外表征。 RecA蛋白-多核苷酸复合物与其他蛋白的相互作用 将研究细胞蛋白，特别是Gly 204的作用 在莱克萨蛋白质结合将被检查。 最后，RecA样活性在同源重组中的作用， DNA修复将通过构建缺乏 编码这种功能的基因。 这些实验将提供 对欧洲大陆重组机制的进一步认识 细胞

The goals of this research proposal are to elucidate and characterize fundamental interactions that are important for the activities of the RecA protein of Escherichia coli. This protein is required for homology dependent recombination, for regulating a set of genes that participate in a DNA damage stress response (SOS response) and for participating directly in the biochemical process leading to mutagenesis. The basic interactions that will be investigated are those involved in i) RecA protein subunit recognition, ii) ATP binding and hydrolysis, and iii) recognition and binding to other proteins such as lambdoid repressors, the cellular LexA repressor and the umuD protein which is needed for a major pathway of chemical mutagenesis. The sequence(s) within the first 90 residues of RecA protein required for subunit binding will be investigated using affinity chromatography and fusion proteins between RecA and beta-lactamase. Binding of synthetic peptides corresponding to the regions of interest will be studied. Two substitution mutations (Tyr264-Phe264 and Tyr264-Ser264) in the ATP binding site have been constructed by site-directed mutagenesis and will be fully characterized with respect to ATP hydrolysis, DNA binding and the ability to carry out DNA pairing (annealing, strand transfer). Second site suppressor mutations will be isolated and characterized in order to identify other important elements of the ATP binding domain. In addition, site-directed mutagenesis will be used to alter residues with a conserved sequence in TecA protein that is found in a number of nucleotide binding proteins. The properties of the proteins will be characterized in vitro. The interaction of RecA protein-polynucleotide complexes with other cellular proteins will be investigated, in particular, the role of Gly204 in LexA protein binding will be examined. Finally, the role of RecA-like activity in homologous recombination and DNA repair will be investigated by constructing yeast strains lacking the gene encoding this function. These latter experiments will provide additional insight into the mechanisms of recombination in eurkaryotic cells.