Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage

Magnesium is essential for the catalysis reaction of Escherichia coli prima se, the enzyme synthesizing primer RNA chains for initiation of DNA replica tion. To map the Mg2+ binding site in the catalytic center of primase, we h ave employed the iron cleavage method in which the native bound Mg2+ ions w ere replaced with Fe2+ ions and the protein was then cleaved in the vicinit y of the metal binding site by adding DTT which generated foe hydroxyl radi cals from the bound iron. Three Fe2+ cleavages were generated at sites desi gnated I, II, and In. Adding Mg2+ or Mn2+ ions to the reaction strongly inh ibited Fe2+ cleavage; however, adding Ca2+ or Ba2+ ions had much less effec t. Mapping by chemical cleavage and subsequent site-directed mutagensis dem onstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid resi dues that chelated Mg2+ ions in the catalytic center of primase. Cleavage d ata suggested that binding to D345 is significantly stronger than to D347 a nd somewhat stronger than to D269.