Roles of divalent metal ions in flap endonuclease-substrate interactions

• Published in: Nature structural & molecular biology Vol. 11; no. 5; pp. 450 - 456
• Main Authors: Sayers, Jon R, Feng, Min, Patel, Dipak, Dervan, Joe J, Ceska, Thomas, Suck, Dietrich, Haq, Ihtshamul
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 01.05.2004
• Subjects: Base Sequence; Binding sites; Biology; Calorimetry; Cations, Divalent - metabolism; Crystal structure; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA polymerase; DNA Primers; Endonucleases - metabolism; Enzyme binding; Enzymes; Hydrolysis; Metal ions; Metals - metabolism; Models, Molecular; Mutation; Nucleases; Phosphate esters; Physiological aspects; Streptococcus infections; Structure; Substrate Specificity; Thermodynamics; United Kingdom; United Kingdom > UK
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/nsmb754

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.