title: Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage.
abstract: Magnesium is essential for the catalysis reaction of Escherichia coli primase, the enzyme synthesizing primer RNA chains for initiation of DNA replication. To map the Mg(2+) binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg(2+) ions were replaced with Fe(2+) ions and the protein was then cleaved in the vicinity of the metal binding site by adding DTT which generated free hydroxyl radicals from the bound iron. Three Fe(2+) cleavages were generated at sites designated I, II, and III. Adding Mg(2+) or Mn(2+) ions to the reaction strongly inhibited Fe(2+) cleavage; however, adding Ca(2+) or Ba(2+) ions had much less effect. Mapping by chemical cleavage and subsequent site-directed mutagensis demonstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid residues that chelated Mg(2+) ions in the catalytic center of primase. Cleavage data suggested that binding to D345 is significantly stronger than to D347 and somewhat stronger than to D269.
authors.affiliation: Biochemistry Department, New York University Medical Center, 550 First Avenue, New York, New York 10016, USA. godsog01@mcrcr6.med.nyu.edu
authors.forename: G N
authors.initials: GN
authors.lastname: Godson
authors.forename: J
authors.initials: J
authors.lastname: Schoenich
authors.forename: W
authors.initials: W
authors.lastname: Sun
authors.forename: A A
authors.initials: AA
authors.lastname: Mustaev
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004229:Dithiothreitol; D004926:Escherichia coli; D007501:Iron / Q000737:chemistry* / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D014357:Trypsin
chemical_list: D002413:Cations, Divalent; D010761:Phosphorus Radioisotopes; D007501:Iron; D019915:DNA Primase; D014357:Trypsin; D008274:Magnesium; D004229:Dithiothreitol