Distinct Roles of Two Mg2+ Binding Sites in Regulation of Murine Flap Endonuclease-1 Activities

Removal of flap DNA intermediates in DNA replication and repair by flap endonuclease-1 (FEN-1) is essential for mammalian genome integrity. Divalent metal ions, Mg2+ or Mn2+, are required for the active center of FEN-1 nucleases. However, it remains unclear as to how Mg2+ stimulates enzymatic activity. In the present study, we systemically characterize the interaction between Mg2+ and murine FEN-1 (mFEN-1). We demonstrate that Mg2+ stimulates mFEN-1 activity at physiological levels but inhibits the activity at concentrations higher than 20 mM. Our data suggest that mFEN-1 exists as a metalloenzyme in physiological conditions and that each enzyme molecule binds two Mg2+ ions. Binding of Mg2+ to the M1 binding site coordinated by the D86 residue cluster enhances mFEN-1's capability of substrate binding, while binding of the metal to the M2 binding site coordinated by the D181 residue cluster induces conformational changes. Both of these steps are needed for catalysis. Weak, nonspecific Mg2+ binding is likely responsible for the enzyme inhibition at high concentrations of the cation. Taken together, our results suggest distinct roles for two Mg2+ binding sites in the regulation of mFEN-1 nuclease activities in a mode different from the “two-metal mechanism”.