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

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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.