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.
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Acknowledgements
We thank J. Grasby for informal discussions and critical reading of the manuscript. This paper is dedicated to the memory of A. Jones. The support of The Wellcome Trust (grant numbers 052123 and 058034) is gratefully acknowledged. The White Rose University Consortium and the BBSRC supported studentships for M.F. and J.J.D., respectively.
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Feng, M., Patel, D., Dervan, J. et al. Roles of divalent metal ions in flap endonuclease–substrate interactions. Nat Struct Mol Biol 11, 450–456 (2004). https://doi.org/10.1038/nsmb754
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DOI: https://doi.org/10.1038/nsmb754
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