Examination of the Role of DNA Polymerase Proofreading in the Mutator Effect of Miscoding tRNAs

We previously described Escherichia coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, «, is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered « subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of mutD alleles. In the present work, we extended the study of specific mutD alleles and constructed 16 altered mutD genes by replacing each aspartic acid codon, in series, with a glycine codon in the dnaQ gene that encodes «. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the mutD gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the « subunit playing a major role in the mutator effect of misreading tRNAs. Mutator genes confer elevated rates of spontaneous mutations. Mutators have been used to help define DNA repair systems and to identify pathways of mutagenesis in prokaryotic as well as eukaryotic cells (24). The findings that certain human inherited cancer susceptibilities result from mutator phenotypes caused by defects in mismatch repair systems (5, 12, 18, 30) have sparked renewed interest in mutators. Defects in the mismatch repair genes lead to a strong mutator phenotype (25), as do combined defects in the GO system that repairs or prevents the incorporation of 7,8-dihydro-8-oxoguanidine in DNA (19, 20, 22). One of the strongest bacterial mutators in Escherichia coli results from a defect in the e subunit of DNA polymerase III (Pol III), which is responsible for the editing function of Pol III (10, 31). The mutD/dnaQ gene encodes the e subunit, and mutations in this gene result in the observed mutator effect. Some of these mutations give very strong effects; an example is the mutD5 allele, which increases the mutation frequency as much as 10 5 -fold in rich medium (6, 9). There are surprisingly few published sequence changes resulting from mutD mutations, and some of the published assignments have been disputed (15, 33). Although mutators have been actively investigated for several decades, new pathways of mutagenesis are still being discovered. We recently described a novel pathway of mutagenesis mediated by mutator tRNAs that are encoded by the E. coli genes mutA and mutC (21, 32). We postulated that the mutator tRNA effect is exerted by mistranslation that generates a mutator polymerase, through an alteration in the e subunit of DNA Pol III (32). The mutator tRNAs are glycine tRNAs with anticodons altered to read the aspartic acid codon and which insert glycine at approximately 1% efficiency. There are 16 aspartic acid codons in the e subunit. In this study, we investigated their importance by replacing each of the 16 codons, in series, with a glycine codon. Three cases resulted in a strong mutator. We also report the construction of an additional mutator tRNA that inserts glycine at histidine codons. We describe the replacement of seven histidine codons in the mutD gene with glycine codons and show that two of the constructed mutants are strong mutators. We discuss these results with regard to the mechanism of the mutator tRNAs and the primary structure of the e subunit.