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

在重组的重要问题中，最热门的问题之一是揭示RecA蛋白的结构与功能之间的关系。为了追求这个问题，在recA基因中不可避免地要有合适的突变体。我们了解到，在表型上有许多有趣的recA突变体，俄罗斯圣彼得堡彼得堡核物理研究所的Lanzov博士，他不熟悉RecA蛋白的理化分析，所以我们同意利用他有用的recA突变体进行合作。此外，他还收集了大量各种嗜热菌，我们还同意从嗜热细菌中分离合适的recA基因，以提供热稳定的RecA蛋白，从而促进物理化学分析。Lanzov博士和他的同事来到我们的实验室，在这两年里进行了联合工作，并获得了以下结果。RecA 2278- 5是具有两个氨基酸取代的突变RecA蛋白，Gly 278被Thr取代，Val 275被Phe取代，该突变体在42*C的基因重组和SOS修复中有缺陷。我们比较了RecA 2278-RecA 2278 -5蛋白的热敏性多重缺陷表明RecA蛋白的C-亚结构域的结构稳定性对于RecA-ATP-ATP结合是必需的。ssDNA螺旋丝的形成是细菌同源重组的第一步。成功分离了一株嗜热菌的recA基因通过PCR方法，根据RecA蛋白ATP结合域的一个共同氨基酸序列设计引物，核苷酸序列分析表明，密码子中第三个核苷酸大部分为G或C，因此DNA富含GC，热稳定。这种特性对于嗜热菌来说是相当合理的。

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.