Efficient Strand Transfer by the RadA Recombinase from the Hyperthermophilic Archaeon Desulfurococcus amylolyticus

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65°C (catalytic rate constant of 1.2 to 2.5 min(−1) at 80 to 95°C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90°C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92°C).

我们对解淀粉脱硫球菌（Desulfurococcus amylolyticus，一种嗜热古菌（hyperthermophilic archaeon））中被推测介导同源重组（homologous recombination）的radA基因完成了克隆、测序，并在大肠杆菌（Escherichia coli）细胞中实现了过表达。该基因编码产物RadA的推导氨基酸序列，与人Rad51蛋白的同源性达65%，高于其与大肠杆菌RecA蛋白的35%同源性。经高度纯化的RadA蛋白可在65℃以上温度下专一催化单链DNA依赖的ATP水解（ATP hydrolysis）反应，该反应可用于监测突触前重组复合物的形成；在80~95℃条件下，其催化速率常数为1.2~2.5 min⁻¹。仅RadA单一蛋白即可在80~90℃（即接近DNA解链温度的温度区间）内高效促进链交换（strand exchange）反应。值得注意的是，ATP水解与链交换反应在该宿主的最适生长温度（90~92℃）下均展现出极高的催化效率。