Identification of the acidic residues in the active site of DNA polymerase III.
The mechanism of nucleotide addition by DNA polymerases involves two metal ions that are coordinated in the active site by conserved acidic residues. The three acidic residues that chelate Mg2+ in the active site of Escherichia coli DNA polymerase III have been identified as Asp401, Asp403, and Asp555 by site-directed mutagenesis. Candidates for mutagenesis were initially chosen based on absolute conservation of acidic residues in an alignment of more than 20 diverse DnaE sequences. Conservative Asp to Glu mutations at positions 401 and 403 reduced the activities of the mutant polymerases 2000 and 333-fold, respectively, from that of the wild-type. The third carboxylate was identified by a series of mutations for each critical candidate. With the exception of Glu, all of the mutations at Asp555 led to severely diminished polymerase activity, while each of the other candidates exhibited several relatively active mutant polymerases. Moreover, only the identified active site mutant polymerases displayed a significant enhancement of activity in Mn2+ compared with Mg2+. These data suggest a direct involvement of the mutated amino acid in metal ion binding.
Pritchard
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AE
Department of Biochemistry and Molecular Genetics B-121, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO, 80262, USA.
McHenry
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CS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics*; D004258:DNA Polymerase III / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D016415:Sequence Alignment
D001426:Bacterial Proteins; D008345:Manganese; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D008274:Magnesium