College of Environmental Science and Engineering, Yangzhou University, Yangzhou, China.
Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, Lianyungang, 222005, China.
World J Microbiol Biotechnol. 2023 Feb 8;39(4):90. doi: 10.1007/s11274-023-03526-2.
Endonuclease V (EndoV), which is widespread in bacteria, eukarya and Archaea, can cleave hypoxanthine (Hx)-containing DNA or RNA strand, and play an essential role in Hx repair. However, our understanding on archaeal EndoV's function remains incomplete. The model archaeon Sulfolobus islandicus REY15A encodes a putative EndoV protein (Sis-EndoV). Herein, we probed the biochemical characteristics of Sis-EndoV and dissected the roles of its seven conserved residues. Our biochemical data demonstrate that Sis-EndoV displays maximum cleavage efficiency at above 60 °C and at pH 7.0-9.0, and the enzyme activity is dependent on a divalent metal ion, among which Mg is optimal. Importantly, we first measured the activation energy for cleaving Hx-containing ssDNA by Sis-EndoV to be 9.6 ± 0.8 kcal/mol by kinetic analyses, suggesting that chemical catalysis might be a rate-limiting step for catalysis. Mutational analyses show that residue D38 in Sis-EndoV is essential for catalysis, but has no role in DNA binding. Furthermore, we first revealed that residues Y41 and D189 in Sis-EndoV are involved in both DNA cleavage and DNA binding, but residues F77, H103, K156 and F161 are only responsible for DNA binding.
内切核酸酶 V（EndoV）广泛存在于细菌、真核生物和古菌中，能够切割含有次黄嘌呤（Hx）的 DNA 或 RNA 链，在 Hx 修复中发挥重要作用。然而，我们对内切核酸酶 V 的功能的理解还不完全。模式古菌 Sulfolobus islandicus REY15A 编码一种假定的内切核酸酶 V 蛋白（Sis-EndoV）。在此，我们研究了 Sis-EndoV 的生化特性，并剖析了其七个保守残基的作用。我们的生化数据表明，Sis-EndoV 在 60°C 以上和 pH7.0-9.0 时显示出最大的切割效率，并且酶活性依赖于二价金属离子，其中 Mg 是最佳的。重要的是，我们首次通过动力学分析测量了 Sis-EndoV 切割含 Hx 的 ssDNA 的活化能为 9.6±0.8 kcal/mol，表明化学催化可能是催化的限速步骤。突变分析表明，Sis-EndoV 中的残基 D38 对于催化是必需的，但对于 DNA 结合没有作用。此外，我们首次揭示了 Sis-EndoV 中的残基 Y41 和 D189 既参与 DNA 切割又参与 DNA 结合，但残基 F77、H103、K156 和 F161 仅负责 DNA 结合。
