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

在重组的重要问题中，揭示RecA蛋白的结构与功能之间的关系是目前研究的热点之一。为了解决这个问题，在recA基因中寻找合适的突变体是不可避免的。我们了解到，在表型上有许多有趣的recA突变体，俄罗斯圣彼得堡核物理研究所的lanzov博士对recA蛋白的理化分析并不熟悉。因此，我们同意利用他有用的recA突变体进行联合研究。此外，他还收集了大量的各种嗜热细菌，这些细菌的蛋白质在物理化学分析的恶劣条件下足够稳定。我们还同意从一种嗜热细菌中分离出合适的recA基因，以提供热稳定的recA蛋白，以便于物理化学分析。lanzov博士和他的同事来到我们的实验室，共同进行了两年的工作，得到了以下结果：RecA2278-5是一种突变的RecA蛋白，在RecA c端亚结构域的a-螺旋H上有两个氨基酸取代，Gly278被Thr取代，Val275被Phe取代。该突变体在42*C时存在基因重组和sos修复缺陷。我们比较了RecA2278-5与野生型RecA蛋白在32 / 42 / cc条件下的生化活性。RecA2278-5蛋白的热敏性多重缺陷表明，RecA蛋白c亚结构域的结构稳定性对于细菌中同源重组的第一步——RecA- atp - ssdna螺旋细丝的形成是必要的。采用PCR方法成功分离了一种嗜热细菌的recA基因，并根据recA蛋白ATP结合域的共同氨基酸序列设计引物。核苷酸序列分析表明，密码子的第三个核苷酸大部分是G或c，因此DNA是富含GC的，并且热稳定。这种特性对嗜热细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.