RECA PROTEIN STRUCTURE, FUNCTION, AND CONTROL

这项研究提案的目标是阐明和描述 对RecA的活动至关重要的基本相互作用 大肠杆菌的蛋白质。这种蛋白质是同源所必需的 依赖重组，用于调节一组参与 DNA损伤应激反应(SOS反应)和直接参与 在导致突变的生化过程中。基本的交互作用 将被调查的是涉及I)RecA蛋白亚基的那些 识别，ii)ATP结合和水解，以及iii)识别和 与其他蛋白质结合，如Lambdoid抑制子，细胞LexA 抑制子和umuD蛋白，这是一个主要的途径 化学诱变。 RECA蛋白前90个残基内的序列(S) 亚基结合将使用亲和层析和 RecA与β-内酰胺酶的融合蛋白。合成纤维的粘合作用 将研究与感兴趣区域相对应的多肽。 ATP中的两个替换突变(Tyr264-Phe264和Tyr264-Ser264) 通过定点突变构建了结合位点，并将 在ATP水解、DNA结合和 能够进行DNA配对(退火、链转移)。第二个站点 将分离和鉴定抑制子突变，以便 确定ATP结合域的其他重要元件。此外, 定点突变将被用于改变保守的残基 在许多核苷酸结合中发现的TecA蛋白的序列 蛋白质。这些蛋白质的性质将在体外进行表征。 RecA蛋白-多核苷酸复合体与其他蛋白质的相互作用 将研究细胞蛋白质，特别是Gly204的作用 在LexA中，将检查蛋白质结合情况。 最后，RecA样活性在同源重组和 DNA修复的研究将通过构建缺乏 编码这一功能的基因。后面的这些实验将提供 对真核生物中重组机制的再认识 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.