The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'-+ 5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacl gene of E. coli. The mutDS mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacI') and constitutive lacO (acOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacFd mutations revealed that transitions occur more frequently than transversions. A. T->G C and G* C--A-T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A T--TT A events were the most common, A *T -C C G substitutions were rare, and G *C->C G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered,' sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G* C sequences. An extraordinary A. T -> G C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutDS mutation frequency of this single base pair was calculated to be 1.2 x 10'.The high fidelity of DNA replication in Escherichia coli is under genetic control. One gene whose product is essential for accurate DNA replication is mutD (dnaQ), which is located at about 5 min on the E. coli genetic map and is closely linked to the structural gene for RNase H, rnh (10,22,23). Two well-characterized mutant alleles of this gene, the dominant'mutDS and the recessive dnaQ49, are strong mutators and raise spontaneous mutation frequencies to 103 to 105 times wild-type levels (10,23,29). The mutDS allele is conditional, with its phenotype dependent upon growth conditions (10). In rich medium such as L-broth or in minimal medium supplemented with thymidine, mutDS strains display extremely high mutation frequencies, whereas in minimal medium without thymidine, mutation frequencies are much lower (50 to 100 times wild-type frequencies). While the nature of this physiological conditionality remains incompletely understood, it has been shown that the thymidine effector which enhances the mutagenic activity of mutDS strains must be phosphorylated (14).The mutD gene product is a 25 to 28-kilodalton protein (9, 22, 27) which is also produced by mutDS mutator strains (9,29). Since 'the dominant mutDS allele 'exhibits negative complementation in mutDS/mut+ merodiploids, it has been suggested that the mutD protein is part of a multimeric structure which could be at the replication fork (9 exonuclease (proofreading) activity (11,13) and that the mutD gene product is the epsilon 'subunit of DNA polymerase III ...