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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 Mg2+ and Mn2+ 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. Copyright © 2022 Elsevier Ltd. All rights reserved.

Citation

Tan Lin, Likui Zhang, Donghao Jiang, Leilei Wu, Kaige Chen, Li Li, Cuili Jin, Zheng Li, Philippe Oger. Biochemical characterization and mutational analysis of a novel flap endonuclease 1 from Thermococcus barophilus Ch5. The international journal of biochemistry & cell biology. 2022 Feb;143:106154

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PMID: 34990837

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