National Center for Biotechnology, Nur-Sultan 010000, Kazakhstan; L. N. Gumilyov Eurasian National University, Nur-Sultan 010000, Kazakhstan.
National Center for Biotechnology, Nur-Sultan 010000, Kazakhstan.
DNA Repair (Amst). 2022 Nov;119:103390. doi: 10.1016/j.dnarep.2022.103390. Epub 2022 Aug 27.
Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Complete genome analysis of Staphylococcus aureus identified a single AP endonuclease, SaNfo, which is a member of the endonuclease IV family exemplified by Escherichia coli Nfo. At present, it remains unknown whether SaNfo possesses DNA repair activities similar to its counterparts from E. coli and other bacteria. Here, we report that the purified SaNfo protein contains efficient AP endonuclease and nucleotide incision repair (NIR) activities. Optimal reaction conditions for SaNfo-catalysed AP endonuclease activity are high ionic strength and Mn concentration, pH in range 7.5-9.0 and the temperature optimum of 37-45 °C. Cell-free extracts of S. aureus exhibited efficient AP site cleavage and NIR activities. Heterologous expression of SaNfo strongly reduces the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and HO. Site-directed mutagenesis showed that the Glu258 residue is critical for the SaNfo enzyme function. The AP endonuclease but not the NIR activity of SaNfo were stimulated by the β-clamp (SaDnaN dimer), suggesting that it might participate in the organization of BER in S. aureus. Overall, our data confirm that the activity, substrate specificity and in vivo functionality of S. aureus Nfo are consistent with this protein being the major AP endonuclease for the repair of DNA damage generated by endogenous and host-imposed factors.
无碱基（AP）内切酶是参与碱基切除修复和 DNA 链断裂修复的关键酶。金黄色葡萄球菌的全基因组分析鉴定出一种单一的 AP 内切酶 SaNfo，它是内切酶 IV 家族的成员，以大肠杆菌 Nfo 为代表。目前，尚不清楚 SaNfo 是否具有与大肠杆菌和其他细菌的同源物相似的 DNA 修复活性。在这里，我们报告纯化的 SaNfo 蛋白具有有效的 AP 内切酶和核苷酸切口修复（NIR）活性。SaNfo 催化的 AP 内切酶活性的最佳反应条件是高离子强度和 Mn 浓度、pH 值在 7.5-9.0 范围内以及 37-45°C 的最适温度。金黄色葡萄球菌的无细胞提取物表现出有效的 AP 位点切割和 NIR 活性。SaNfo 的异源表达强烈降低了 AP 内切酶缺陷型大肠杆菌 xth nfo 菌株对甲基甲磺酸和 HO 的敏感性。定点突变显示 Glu258 残基对于 SaNfo 酶的功能至关重要。SaNfo 的 AP 内切酶但不是 NIR 活性被 β-夹（SaDnaN 二聚体）刺激，表明它可能参与金黄色葡萄球菌中 BER 的组织。总的来说，我们的数据证实了金黄色葡萄球菌 Nfo 的活性、底物特异性和体内功能与该蛋白作为修复内源性和宿主诱导因素产生的 DNA 损伤的主要 AP 内切酶一致。
