College of Environmental Science and Engineering, Marine Science & Technology Institute, Yangzhou University, China.
College of Environmental Science and Engineering, Marine Science & Technology Institute, Yangzhou University, China.
Int J Biochem Cell Biol. 2022 Feb;143:106154. doi: 10.1016/j.biocel.2021.106154. Epub 2022 Jan 3.
Flap endonuclease 1 (FEN1) plays important roles in DNA replication, repair and recombination. Herein, we report biochemical characteristics and catalytic mechanism of a novel FEN1 from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tb-FEN1). As expected, the recombinant Tb-FEN1 can cleave 5'-flap DNA. However, the enzyme has no activity on cleaving pseudo Y DNA, which sharply contrasts with other archaeal and eukaryotic FEN1 homologs. Tb-FEN1 retains 24% relative activity after heating at 100 °C for 20 min, demonstrating that it is the most thermostable among all reported FEN1 proteins. The enzyme displays maximal activity in a wide range of pH from 7.0 to 9.5. The Tb-FEN1 activity is dependent on a divalent metal ion, among which Mg and Mn are optimal. Enzyme activity is inhibited by NaCl. Kinetic analyzes estimated that an activation energy for removal of 5'-flap from DNA by Tb-FEN1 was 35.7 ± 4.3 kcal/mol, which is the first report on energy barrier for excising 5'-flap from DNA by a FEN1 enzyme. Mutational studies demonstrate that the K87A, R94A and E154A amino acid substitutions abolish cleavage activity and reduce 5'-flap DNA binding efficiencies, suggesting that residues K87, R94, and E154 in Tb-FEN1 are essential for catalysis and DNA binding as well. Overall, Tb-FEN1 is an extremely thermostable endonuclease with unusual features.
Flapendonuclease 1（FEN1）在 DNA 复制、修复和重组中发挥着重要作用。在此，我们报道了来自嗜热古菌 Thermococcus barophilus Ch5 的一种新型 FEN1（Tb-FEN1）的生化特性和催化机制。正如预期的那样，重组 Tb-FEN1 可以切割 5'-flap DNA。然而，该酶对切割假 Y DNA 没有活性，这与其他古菌和真核 FEN1 同源物形成鲜明对比。Tb-FEN1 在 100°C 加热 20 分钟后仍保留 24%的相对活性，表明它是所有报道的 FEN1 蛋白中最耐热的。该酶在 pH7.0 至 9.5 的较宽范围内显示出最大的活性。Tb-FEN1 的活性依赖于二价金属离子，其中 Mg 和 Mn 是最佳的。酶活性受 NaCl 抑制。动力学分析估计，Tb-FEN1 从 DNA 上切除 5'-flap 的活化能为 35.7±4.3 kcal/mol，这是首次报道 FEN1 酶从 DNA 上切除 5'-flap 的能量障碍。突变研究表明，K87A、R94A 和 E154A 氨基酸取代会使切割活性丧失，并降低 5'-flap DNA 的结合效率，表明 Tb-FEN1 中的 K87、R94 和 E154 残基对催化和 DNA 结合也是必不可少的。总的来说，Tb-FEN1 是一种具有特殊特性的极其耐热的内切核酸酶。
