Identification of Escherichia coli dnaE ( polC ) Mutants with Altered Sensitivity to 2′,3′-Dideoxyadenosine

Abstract

ABSTRACT Bacteria with reduced DNA polymerase I activity have increased sensitivity to killing by chain-terminating nucleotides (S. A. Rashbaum and N. R. Cozzarelli, Nature 264:679–680, 1976). We have used this observation as the basis of a genetic strategy to identify mutations in the dnaE ( polC ) gene of Escherichia coli that alter sensitivity to 2′,3′-dideoxyadenosine (ddA). Two dnaE ( polC ) mutant strains with increased sensitivity to ddA and one strain with increased resistance were isolated and characterized. The mutant phenotypes are due to single amino acid substitutions in the α subunit, the protein product of the dnaE ( polC ) gene. Increased sensitivity to ddA is produced by the L329F and H417Y substitutions, and increased resistance is produced by the G365S substitution. The L329F and H417Y substitutions also reduce the accuracy of DNA replication (the mutator phenotype), while the G365S substitution increases accuracy (the antimutator phenotype). All of the amino acid substitutions are in conserved regions near essential aspartate residues. These results prove the effectiveness of the genetic strategy in identifying informative dnaE ( polC ) mutations that can be used to elucidate the molecular basis of nucleotide interactions in the α subunit of the DNA polymerase III holoenzyme.