Download MendeleyRoles of Divalent Metal Ions in Flap Endonuclease-Substrate Interactions
Feng, M., Patel, D., Dervan, J., Ceska, T.A., Suck, D., Haq, I., Sayers, J.R.(2004) Nat Struct Mol Biol 11: 450
- PubMed: 15077103
- DOI: https://doi.org/10.1038/nsmb754
- Primary Citation of Related Structures:
1UT5, 1UT8 - PubMed Abstract:
Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete loss of catalytic activity. Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity. Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II. Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor. The data suggest that a novel two-metal mechanism operates in the FEN-catalyzed exonucleolytic reaction. These results raise the possibility that local concentrations of free cofactor could influence the endo- or exonucleolytic pathway in vivo.
Organizational Affiliation:
University of Sheffield School of Medicine and Biomedical Science, Division of Genomic Medicine, Beech Hill Road, Sheffield, S10 2RX, UK.
