Roles of divalent metal ions in flap endonuclease-substrate interactions.
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.
Feng
Min
M
University of Sheffield School of Medicine and Biomedical Science, Division of Genomic Medicine, Beech Hill Road, Sheffield, S10 2RX, UK.
Patel
Dipak
D
Dervan
Joe J
JJ
Ceska
Thomas
T
Suck
Dietrich
D
Haq
Ihtshamul
I
Sayers
Jon R
JR
D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004720:Endonucleases / Q000378:metabolism*; D006868:Hydrolysis; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D013379:Substrate Specificity; D013816:Thermodynamics
D002413:Cations, Divalent; D017931:DNA Primers; D008670:Metals; D004247:DNA; D004720:Endonucleases