Enzymatic characterization of Sulfolobus acidocaldarius Saci0542 nuclease belonging to DHH superfamily

Abstract:

[Objective] To provide biochemical support for studying the potential function during DNA repair via systematically characterizing the enzymatic properties of the DHH superfamily nuclease (Saci0542) of Sulfolobus acidocaldarius. [Methods] Saci0542 was recombinantly expressed in Escherichia coli and then purified by affinity chromatography. The enzymatic properties of Saci0542 were characterized in vitro with fluorescence-labeled oligonucleotides as substrates by urea-denaturing polyacrylamide gel electrophoresis. [Results] The recombinant Saci0542 had a typical 3'-5' exonuclease activity on ssDNA. Its activity was dependent on the divalent metal ion Mn²⁺ and inhibited by the divalent metal ions such as Ca²⁺, Mg²⁺, and Zn²⁺. Saci0542 showed high activity in a wide range of pH 5.5-10. NaCl above 50 mmol/L strongly inhibited the activity of this enzyme, and the optimum reaction temperature was 50-55℃. The terminal phosphate group inhibited the 3'-5' exonuclease activity. [Conclusion] This study confirmed that Saci0542 was a Mn²⁺-dependent 3'-5' exonuclease with the activity similar to that of NrnA nuclease, which may be responsible for DNA repair or RNA degradation and recycling in the cell.