Expression, purification and characterization of RecJ nuclease from Archaeoglobus fulgidus

Abstract:

[Objective] To clone, express and purify the RecJ nuclease gene (AF_0699) from Archaeoglobus fulgidus, identify and analyze its nuclease activity. [Methods] Recombinant RecJ nuclease (AfuRecJ) was expressed in E. coli and purified by affinity chromatography. The enzymatic properties of AfuRecJ nuclease were characterized in vitro using synthesized oligo (deoxy) nucleotides as substrate. [Results] AfuRecJ nuclease has a single-stranded DNA specific 3'–5' exonuclease activity, which depends on the divalent metal ions Mn²⁺ or Mg²⁺, and the catalytic efficiency of Mn²⁺ is significantly higher than that of Mg²⁺. The optimal reaction temperature is between 55 and 65 ℃. The presence of high concentration of NaCl decreases the exonuclease activity of AfuRecJ, and cleavage percentage greatly reduces at 200 mmol/L of NaCl. AfuRecJ shows 3'–5' exonuclease activity on single-stranded RNA, higher than that of single-stranded DNA. The 3' terminal phosphate group inhibits the AfuRecJ nuclease. AfuRecJ has different preferences for substrate length. It can effectively hydrolyze ssRNA≥4 nt, and ssDNA≥12 nt. Moreover, the hydrolysis activity of AfuRecJ on dsDNA with special structure (3' overhang and 3' fork) is similar to that of ssDNA. [Conclusion] The nuclease activity of AfuRecJ depends on the Mn²⁺ and can cleave ssDNA and ssRNA with a preference for RNA substrate.