Molecular evolution of recombinational mechanism

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The RecA protein plays an indispensable role in genetic recombination in eubacteria. Recently, the RAD51 protein, which is homologous to the RecA protein, was found in many eukaryotes. However, functional domains and reaction mechanism of RecA/RAD51 protein remain uncertain. Since a protein from thermoresistant organisms is knows to be very stable and liable to be crystallization. Then we collected many thermoresistant microorganisms from hot springs in Russia and selected several bacterial strains, Methanosarcina sp. TS-2, Halococcus turomenicus B1734, Desulfurococcus amynoliticus, Halobacterium distributum B1733, B1739, adequate for biochemical study. Two highly conserved regions of the RecA/RAD51 proteins were chosen to synthesize oligonucleotide primers for PCR.The DNA fragments ware amplified in genomic DNAa by PCR.Using these fragments as a probe, we screened a gene bank of its genomic DNA.From the positive plaques, clones carrying the recA/RAD51 gene were selected by Southern hybridization and their nucleotide sequences ware determined. Comparison of primary structures of RecA/RAD51 proteins of archaea, eubacteria, and eukaryotes suggested that the archeal RecA/RAD51 protein may be an ancestral prototype of eubacterial and eularyotic enzymes. We also cloned the recA gene of another thermophilic bacterium, Thermus thermophilus HB8. Its gene product had fundamental activities for RecA protein. As T.thermophilus RecA protein was very stable, we succeeded in crystallization of this protein both in isolation and in the presence of DNA.Furthermore, we made truncated mutant of T.thermophilus RecA protein to prevent its aggregation at high protein concentrations. As this truncated protein was also stable, it was suitable for NMR measurements for resolving its tertiary structure.