嗜热古菌Sulfolobus acidocaldarius核酸内切酶V的重组表达与酶学特征

Recombinant expression and enzymatic characterization of endonuclease V from Sulfolobus acidocaldarius

摘要:

[目的] 表达纯化嗜酸嗜热硫化叶菌（Sulfolobus acidocaldarius）的核酸内切酶V （Saci_0544），对其核酸内切酶活性及酶学特征进行探究。[方法] 将Sulfolobus acidocaldarius核酸内切酶V （SacEndoV）在大肠杆菌中进行重组表达，经亲和层析纯化得到目标蛋白；利用带有不同类型损伤的寡核苷酸作为底物，结合变性聚丙烯酰胺凝胶电泳技术，鉴定SacEndoV对相应损伤寡核苷酸底物的剪切活性。[结果] SacEndoV特异性剪切含脱氧肌苷（Deoxyinosine）的损伤DNA底物，明显偏好单链DNA底物。SacEndoV在70-95℃温度范围内酶活性高，酶活性依赖于二价金属离子，Mg²⁺为最佳辅助离子，其最佳反应pH为7.5-8.0，高于200 mmol/L的NaCl会明显抑制其剪切活性。损伤DNA中脱氧肌苷3'端相邻的脱氧核糖核苷酸的结构完整性对于SacEndoV识别并剪切相应底物具有重要影响，脱氧肌苷3'端无碱基位点的存在使得SacEndoV不能够切断损伤DNA。此外，经测定SacEndoV对于含肌苷的损伤RNA底物具有剪切活性。[结论] 本研究证实SacEndoV是一种典型的核酸内切酶V，对含脱氧肌苷的损伤DNA具有特异性的内切酶活性，推测其在Sulfolobus acidocaldarius体内参与脱氧肌苷的切除修复。

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.