title: Distinct roles of two Mg2+ binding sites in regulation of murine flap endonuclease-1 activities.
abstract: 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, Mg(2+) or Mn(2+), are required for the active center of FEN-1 nucleases. However, it remains unclear as to how Mg(2+) stimulates enzymatic activity. In the present study, we systemically characterize the interaction between Mg(2+) and murine FEN-1 (mFEN-1). We demonstrate that Mg(2+) 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 Mg(2+) ions. Binding of Mg(2+) 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 Mg(2+) binding is likely responsible for the enzyme inhibition at high concentrations of the cation. Taken together, our results suggest distinct roles for two Mg(2+) binding sites in the regulation of mFEN-1 nuclease activities in a mode different from the "two-metal mechanism".
authors.affiliation: Division of Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zheng
authors.forename: Mei
authors.initials: M
authors.lastname: Li
authors.forename: Jixiu
authors.initials: J
authors.lastname: Shan
authors.forename: Ramaswamy
authors.initials: R
authors.lastname: Krishnamoorthi
authors.forename: Binghui
authors.initials: B
authors.lastname: Shen
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D045585:Flap Endonucleases; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry
chemical_list: D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; D014357:Trypsin; D008274:Magnesium