Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe.
The Escherichia coli dnaQ gene encodes the proofreading 3' exonuclease (epsilon subunit) of DNA polymerase III holoenzyme and is a critical determinant of chromosomal replication fidelity. We constructed by site-specific mutagenesis a mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12-->Ala and Glu-14-->Ala) in the Exo I motif, which, by analogy to other proofreading exonucleases, is essential for the catalytic activity. When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, still binds to the polymerase subunit (alpha subunit or dnaE gene product). When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells became inviable. However, viable dnaQ926 strains could be obtained if they contained one of the dnaE alleles previously characterized in our laboratory as antimutator alleles or if it carried a multicopy plasmid containing the E. coli mutL+ gene. These results suggest that loss of proofreading exonuclease activity in dnaQ926 is lethal due to excessive error rates (error catastrophe). Error catastrophe results from both the loss of proofreading and the subsequent saturation of DNA mismatch repair. The probability of lethality by excessive mutation is supported by calculations estimating the number of inactivating mutations in essential genes per chromosome replication.
Fijalkowska
I J
IJ
Laboratory of Molecualr Genetics, NationalInstitute of Enviromental Health Sciences, Research Triangle Park, NC 27709, USA.
Schaaper
R M
RM
D000595:Amino Acid Sequence; D002876:Chromosomes, Bacterial; D004258:DNA Polymerase III / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004171:Diploidy; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D005798:Genes, Bacterial; D005838:Genotype; D006238:Haploidy; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014780:Viruses / Q000201:enzymology / Q000235:genetics
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D004258:DNA Polymerase III; C468316:dnaQ protein, E coli; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V