Sulfolobus acidocaldarius DHH超家族核酸酶Saci0542的酶学特征研究

Enzymatic characterization of Sulfolobus acidocaldarius Saci0542 nuclease belonging to DHH superfamily

摘要:

【目的】以嗜酸嗜热硫化叶菌Sulfolobus acidocaldarius的DHH超家族核酸酶（Saci0542）为例，研究其核酸外切酶活性特点，为阐明其在DNA代谢中的具体功能提供生化基础。【方法】将嗜酸嗜热硫化叶菌DHH超家族核酸酶Saci0542基因在大肠杆菌中重组表达，经亲和层析纯化得到电泳纯的重组蛋白；利用荧光标记的寡核苷酸作为底物，用尿素变性聚丙烯酰胺凝胶电泳技术，鉴定Saci0542的酶学特征。【结果】重组表达的DHH超家族核酸酶Saci0542具有典型的单链核酸特异性的3'-5'外切酶活性。进一步酶学特征表征结果如下：酶活性依赖于二价金属离子Mn²⁺，而Ca²⁺、Mg²⁺、Zn²⁺等二价金属离子对活性没有明显的促进作用；Saci0542在pH 5.5–10的广泛范围内均表现出较高酶活性；高于200 mmol/L的NaCl强烈抑制酶活性；最适反应温度为50–55℃；末端磷酸基团抑制3'-5'外切酶活性。【结论】本研究证实，Saci0542是一种Mn²⁺依赖型3'-5'外切酶，酶活性与NrnA核酸酶相似，可能在细胞内负责DNA修复或RNA的降解再循环利用。

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.