Identification of the Magnesium Ion Binding Site in the Catalytic Center of Escherichia coli Primase by Iron Cleavage

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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 Mg2+ binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg2+ ions were replaced with Fe2+ 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 Fe2+ cleavages were generated at sites designated I, II, and III. Adding Mg2+ or Mn2+ ions to the reaction strongly inhibited Fe2+ cleavage; however, adding Ca2+ or Ba2+ 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 Mg2+ 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.