College of Environmental Science and Engineering, Yangzhou University, China.
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong-Chuan Road, Shanghai 200240, China.
Int J Biol Macromol. 2023 Mar 1;230:123222. doi: 10.1016/j.ijbiomac.2023.123222. Epub 2023 Jan 11.
Uracil DNA glycosylase (UDG) can remove uracil from DNA, thus playing an essential role in maintaining genomic stability. Family IV UDG members are mostly widespread in hyperthermophilic Archaea and bacteria. In this work, we characterized the family IV UDG from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A (Sis-UDGIV) biochemically, and dissected the roles of nine conserved residues in uracil excision by mutational analyses. Biochemical data demonstrate that Sis-UDGIV displays maximum efficiency for uracil excision at 50 °C ~ 70 °C and at pH 7.0-9.0. Additionally, the enzyme has displays a weak activity without a divalent metal ion, but maximum activity with Mg. Our mutational analyses show that residues E48 and F55 in Sis-UDGIV are essential for uracil removal, and residues E48, F55, R87, R92 and K146 are responsible for binding DNA. Importantly, we systemically revealed the roles of four conserved cysteine residues C14, C17, C86 and C102 in Sis-UDGIV that are required for being ligands of FeS cluster in maintaining the overall protein conformation and stability by circular dichroism analyses. Overall, our work has provided insights into biochemical function and DNA-binding specificity of archaeal family IV UDGs.
尿嘧啶 DNA 糖基化酶（UDG）可以从 DNA 中去除尿嘧啶，从而在维持基因组稳定性方面发挥重要作用。家族 IV UDG 成员主要广泛存在于高温古菌和细菌中。在这项工作中，我们从高温古菌 Sulfolobus islandicus REY15A（Sis-UDGIV）中对家族 IV UDG 进行了生化特性分析，并通过突变分析解析了 9 个保守残基在尿嘧啶切除中的作用。生化数据表明，Sis-UDGIV 在 50°C~70°C 和 pH7.0-9.0 时显示出最大的尿嘧啶切除效率。此外，该酶在没有二价金属离子的情况下表现出较弱的活性，但在有 Mg 的情况下表现出最大的活性。我们的突变分析表明，Sis-UDGIV 中的残基 E48 和 F55 对于尿嘧啶的去除是必需的，而残基 E48、F55、R87、R92 和 K146 负责与 DNA 结合。重要的是，我们系统地揭示了四个保守半胱氨酸残基 C14、C17、C86 和 C102 在 Sis-UDGIV 中的作用，这些残基是作为 FeS 簇配体所必需的，以维持整体蛋白质构象和稳定性，这是通过圆二色性分析揭示的。总的来说，我们的工作为了解古菌家族 IV UDG 的生化功能和 DNA 结合特异性提供了新的见解。
