Biochemical characterization and mutational analysis of a novel flap endonuclease 1 from Thermococcus barophilus Ch5 - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue International Journal of Biochemistry and Cell Biology Année : 2022

Biochemical characterization and mutational analysis of a novel flap endonuclease 1 from Thermococcus barophilus Ch5

Tan Lin
  • Fonction : Auteur
Likui Zhang
  • Fonction : Auteur
  • PersonId : 1083919
Leilei Wu
  • Fonction : Auteur
Kaige Chen
  • Fonction : Auteur
Li Li
  • Fonction : Auteur
Cuili Jin
  • Fonction : Auteur

Résumé

Flap endonuclease 1 (FEN1) plays important roles in DNA replication, repair, and recombination. Herein, we report the 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 o 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 2+ and Mn 2+ are optimal. Enzyme activity is inhibited by NaCl. Kinetic analyses estimated that the activation energy for the removal of 5′-flap from DNA by Tb-FEN1 was 35.7 ± 4.3 kcal/mol, which is the first report on the 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.
Fichier principal
Vignette du fichier
PO_Revised Manuscript 3 (1).pdf (5.15 Mo) Télécharger le fichier
Origine : Fichiers produits par l'(les) auteur(s)

Dates et versions

hal-03722823 , version 1 (13-07-2022)

Identifiants

Citer

Tan Lin, Likui Zhang, Donghao Jiang, Leilei Wu, Kaige Chen, et al.. Biochemical characterization and mutational analysis of a novel flap endonuclease 1 from Thermococcus barophilus Ch5. International Journal of Biochemistry and Cell Biology, 2022, 143, pp.106154. ⟨10.1016/j.biocel.2021.106154⟩. ⟨hal-03722823⟩
20 Consultations
57 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More