Roles of divalent metal ions in flap endonuclease-substrate interactions
| Autor: | Thomas A. Ceska, Min Feng, Joe J. Dervan, Ihtshamul Haq, Dietrich Suck, Jon R. Sayers, Dipak Patel |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Exonuclease
Models Molecular Stereochemistry Cations Divalent Cofactor Divalent Substrate Specificity chemistry.chemical_compound Endonuclease Structural Biology Flap endonuclease Molecular Biology DNA Primers chemistry.chemical_classification biology Base Sequence Hydrolysis Isothermal titration calorimetry DNA Endonucleases Enzyme chemistry Biochemistry Metals biology.protein Thermodynamics |
| Zdroj: | Nature structuralmolecular biology. 11(5) |
| ISSN: | 1545-9993 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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