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

【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3ʹ-5ʹ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Mn^(2+),而Ca^(2+)、Mg^(2+)、Zn^(2+)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°C;末端磷酸基团抑制3ʹ-5ʹ外切酶活性。【结论】本研究证实,Saci0542是一种Mn^(2+)依赖型3ʹ-5ʹ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。

[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 Mn^(2+)and inhibited by the divalent metal ions such as Ca^(2+),Mg^(2+),and Zn^(2+).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°C.The terminal phosphate group inhibited the 3′-5′exonuclease activity.[Conclusion]This study confirmed that Saci0542 was a Mn^(2+)-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.