Identification of Escherichia coli dnaE (polC) Mutants with altered sensitivity to 2 ',3 '-dideoxyadenosine

Bacteria with reduced DNA polymerase I activity have increased sensitivity to killing by chain-terminating nucleotides (S. A. Rashbaum and N. R. Cozza relli, Nature 264:679-680, 1976). We have used this observation as the basi s of a genetic strategy to identify mutations in the dnaE (polC) gene of Es cherichia 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 phen otypes are due to single amino acid substitutions in the alpha subunit, the protein product of the dnaE (polC) gene. Increased sensitivity to ddA is p roduced by the L329F and H417Y substitutions, and increased resistance is p roduced by the G365S substitution. The L329F and H417Y substitutions also r educe the accuracy of DNA replication (the mutator phenotype), while the G3 65S 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 elucidat e the molecular basis of nucleotide interactions in the a subunit of the DN A polymerase III holoenzyme.