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The Characteristics of Thermo-Stable RecA Protein : The Molecular Mechanisms in Genetic Recombination

热稳定性RecA蛋白的特性：基因重组的分子机制

基本信息

批准号：
05044132
负责人：
OGAWA Hideyuki
金额：
$ 3.2万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

Among important problems in recombination, one of the hottest problems is to reveal a relationship between structure and function of RecA protein.To pursue this problem, it is inevitable to have suitable mutants in the recA gene.We learned that having many interesting recA mutants in phenotype, Dr.Lanzov, St.Petersburg Nuclear Physics Institute in Russia, was not familiar in physico-chemical analysis of RecA protein.Therefore we agreed to do a joint work using his useful recA mutants.Furthermore, he also has a big collection of various kinds of thermophilic bacteria, proteins of which are stable enough for sever conditions during physico-chemical analysis.We also agreed to isolate a suitable recA gene from a thermophilic bacteria to provide a heat stable RecA protein to facilitate physico-chemical analyzes.Dr.Lanzov with his colleagues came to our lab and did the joint works for these two years and obtained following results.RecA2278-5 is a mutant RecA protein bearing two amino acid substitutions, Gly278 by Thr and Val275 by Phe, in the a-helix H of C-terminal sub-domain of the RecA.The mutant is defective in genetic recombination and SOS-repair at 42*C.We compared the biochemical activities of RecA2278-5 with those of a wild type RecA protein at 32ﾟand 42ﾟC.The thermosensitive multiple deficiencies of the RecA2278-5 protein suggest that the structural stability of C-subdomain of the RecA protein is necessary for RecA-ATP-ssDNA helix filament formation that is a primary step of homologous recombination in bacteria.The recA gene of a thermophilic bacteria was successfully isolated by PCR methods, in which the primers were designed after a common amino acid sequence for an ATP binding domain of RecA proteins.Nucleotide sequence analysis revealed that most of the third nucleotide in a codon is G or C.Therefore the DNA is GC rich and heat-stable. This characteristic is quite reasonable for thermophilic bacteria.

在重组的重要问题中，最热门的问题之一就是揭示RecA蛋白的结构和功能之间的关系。为了解决这个问题，在recA基因中找到合适的突变体是不可避免的。我们了解到，俄罗斯圣彼得堡核物理研究所的Lanzov博士在表型上有许多有趣的recA突变体，对RecA蛋白的物理化学分析并不熟悉。因此，我们同意利用他有用的recA突变体进行联合工作。此外，他还收集了大量各种嗜热菌。我们还同意从一株嗜热菌中分离出一个合适的recA基因，以提供一个热稳定的RecA蛋白，以便于进行理化分析。Lanzov博士和他的同事们来到我们的实验室，进行了这两年的联合工作，得到了以下结果。RecA2278-5是一个突变的RecA蛋白，含有两个氨基酸取代，Gly278由Thr取代，Val275由Phe取代，通过比较RecA2278-5和野生型RecA蛋白在32ﾟ和42ﾟC时的生化活性，发现RecA2278-5蛋白对温度敏感的多重缺陷表明，RecA蛋白C-亚区结构稳定是形成RecA-ATP-ssDNA螺旋微丝所必需的，这是细菌同源重组的首要步骤。核苷酸序列分析表明，密码子中的第三个核苷酸大部分是G或C，因此该DNA富含GC且具有热稳定性。这一特性对于嗜热菌来说是相当合理的。