Recombinant expression and enzymatic characterization of endonuclease V from Sulfolobus acidocaldarius

Abstract:

[Objective] To express and purify the endonuclease V (Saci_0544) from Sulfolobus acidocaldarius, identify its endonuclease activity and enzymatic characterization. [Methods] The endonuclease V (SacEndoV) from Sulfolobus acidocaldarius was expressed in E. coli and purified by affinity chromatography; Oligonucleotides with different types of damage were used as substrates to identify the cleavage activity of SacEndoV. [Results] SacEndoV specifically cleaves damaged DNA substrates containing deoxyinosine. Compared with double-stranded DNA substrates, the enzyme has a clear preference for single-stranded DNA substrates in vitro. The enzyme activity of SacEndoV is excellent in the temperature range of 70-95℃. And its enzyme activity depends on the divalent metal ion, Mg²⁺ is the best cofactor. The optimal reaction pH of SacEndoV is 7.5-8.0, and NaCl with a concentration higher than 200 mmol/L will significantly inhibit its cleavage activity. The structural integrity of deoxyribonucleotide adjacent to the 3' end of deoxyinosine in the damaged DNA is of great significance for SacEndoV to recognize and cleave the corresponding substrates. The presence of AP sites at the 3' end of deoxyinosine prevents SacEndoV from cleaving damaged DNA. In addition, it has been determined that SacEndoV has cleavage activity on damaged RNA substrates containing inosine.[Conclusion] This study confirmed that SacEndoV is a typical endonuclease V with substrate specificity for damaged DNA containing deoxyinosine, and participates in the repair of deoxyinosine in Sulfolobus acidocaldarius.