Molecular evolution of recombinational mechanism

RecA蛋白在真细菌的基因重组中起着不可或缺的作用。最近，在许多真核生物中发现了与RecA蛋白同源的RAD 51蛋白。然而，RecA/RAD 51蛋白的功能结构域和反应机制仍不清楚。因为已知来自耐热生物的蛋白质非常稳定并且易于结晶。然后从俄罗斯温泉中采集了大量的耐热微生物，从中筛选出适合于生物化学研究的菌株：甲烷八叠球菌TS-2、土耳其盐球菌B1734、解氨脱硫球菌、分布盐杆菌B1733、B1739。选择RecA/RAD 51蛋白的两个高度保守区合成寡核苷酸引物，用PCR方法从基因组DNA中扩增出DNA片段，以此片段为探针筛选其基因组DNA库，从阳性噬菌斑中筛选出携带recA/RAD 51基因的克隆，并对其核苷酸序列进行测定。RecA/RAD 51蛋白的古细菌，真细菌和真核生物的一级结构的比较表明，古细菌RecA/RAD 51蛋白可能是真细菌和真核生物酶的祖先原型。我们还克隆了另一种嗜热菌Thermus thermophilus HB 8的recA基因。其基因产物具有RecA蛋白的基本活性。由于嗜热嗜热菌RecA蛋白非常稳定，我们成功地在分离和存在DNA的情况下结晶了该蛋白，并且制备了嗜热嗜热菌RecA蛋白的截短突变体以防止其在高蛋白浓度下聚集。由于这种截短的蛋白质也是稳定的，它适合于NMR测量来解析其三级结构。

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.