Functional domains of E.coli RecA protein for D-loop formation.

大肠杆菌RecA蛋白的体外反应研究对于理解同源DNA重组的分子机制具有重要的意义。在上述课题资助的研究中，我们集中精力分析RecA蛋白的分子结构，以阐明RecA蛋白在链转移反应中的分子机制。在1988和1989财政年度，我们取得了如下结果。1)通过对定点突变产生的几种改变的phi 8 O cI阻遏蛋白的分析，发现RecA蛋白不参与阻遏物蛋白水解的催化机制。RecA蛋白通过改变其构象增强阻遏物的自切割活性，表明RecA蛋白具有蛋白酶的任何功能性供体。2)从C端缺失25个氨基酸残基的RecA 5327蛋白与单链和双链DNA的结合亲和力均高于野生型RecA蛋白。其他证据支持的C-末端区域负调控RecA蛋白的DNA结合活性。3)从40街开始。到60号。N-末端富含疏水性氨基酸残基的残基起RecA-RecA相互作用的作用。在此基础上，我们构建了RecA蛋白的头对头二聚体模型，该模型假定RecA蛋白含有一个DNA结合结构域、两个RecA-RecA相互作用结构域和ATP酶结构域，以解释RecA蛋白形成D环的分子机制。

The study of in vitro reaction of the RecA protein of Escherichia coli has been playing a frontier role for understanding the molecular mechanism of homologous DNA recombination. In the study funded under the title described above, we focused our efforts on the analyses of molecular structure of the RecA protein to elucidate the molecular mechanism of RecA protein in the strand transfer reaction. In the fiscal years of 1988 and 1989, we obtained the results as described below. 1) Through the analyses of several altered phi8O cI repressor proteins generated by the site directed mutagenesis, it was revealed that the RecA protein does not involved in the catalytic mechanism of the proteolysis of the repressor. The RecA protein enhances the autocleavage activity of the repressor by changing its conformation, suggesting that the RecA protein has any functional donmains for protease. 2) The RecA5327 protein which is deleted 25 amino acids residues from its C-terminal bound to both of single and double stranded DNA with higher affinities than the wild-type RecA protein. Other evidences supporting that the C-terminal region negatively regulates the DNA binding activity of RecA protein were obtained. 3) The region from 40th. to 60th. residues from N-terminal where is rich in hydrophobic amino acid residues functions for RecA-RecA interaction. From these results, we constructed the Head to Head Dimer Model of RecA protein, in which the RecA protein is postulated to contain one DNA binding domain, two domains for RecA-RecA interactions, ATPase domain for expaining the molecular mechanism of RecA protein in forming D-loop.