Translational stress-induced mutagenesis (TSM) refers to the elevated mutagenesis observed in Escherichia coli cells in which mistranslation has been increased as a result of mutations in tRNA genes (such as mutA) or by exposure to streptomycin. TSM does not require lexA-regulated SOS functions but is suppressed in cells defective for homologous recombination genes. Crude cell-free extracts from TSM-induced E. coli strains express an error-prone DNA polymerase. To determine whether DNA polymerase III is involved in the TSM phenotype, we first asked if the phenotype is expressed in cells defective for all four of the non-replicative DNA polymerases, namely polymerase I, II, IV, and V. By using a colony papillation assay based on the reversion of a lacZ mutant, we show that the TSM phenotype is expressed in such cells. Second, we asked if pol III from TSM-induced cells is error-prone. By purifying DNA polymerase III* from TSM-induced and control cells, and by testing its fidelity on templates bearing 3,N 4 -ethenocytosine (a mutagenic DNA lesion), as well as on undamaged DNA templates, we show here that polymerase III* purified from mutA cells is error-prone as compared with that from control cells. These findings suggest that DNA polymerase III is modified in TSMinduced cells.Autonomous organisms normally replicate its DNA accurately, but the fidelity of replication can be transiently decreased in response to environmental and physiological stimuli through a number of pathways (1). Although the Escherichia coli SOS response represents the best-described transient mutator response (2), emerging evidence indicates the existence of multiple inducible mutagenic pathways in E. coli (3). One especially intriguing pathway is provoked by increased translational errors resulting from mutations in tRNA genes (4, 5), in genes specifying tRNA-modification enzymes (6), or from exposure to streptomycin, an antibiotic that promotes mistranslation (7). This pathway, dubbed translational stress-induced mutagenesis (TSM) 1 (3), does not require the induction of lexA/ recA-regulated SOS genes and is suppressed in cells defective for RecABC/RuvABC-dependent homologous recombination (5, 7-9). Available genetic (5, 7-9) and biochemical (10) evidence suggests that the TSM phenotype results from error-prone DNA replication rather than from defective DNA repair. On the basis of the effect of elimination of individual genes encoding "non-replicative" polymerases, we previously proposed that either DNA polymerase III or an unidentified new DNA polymerase is responsible for error-prone replication in TSM-induced cells. DNA polymerase III holoenzyme (pol III HE) accounts for more than 90% of cellular DNA synthesis (11, 12) and is also required for the major post-replicative mismatch correction pathway (13, 14). pol III HE was shown to effectively carry out translesion DNA synthesis past abasic sites, mostly producing Ϫ1-bp deletions (15), as contrasted to translesion synthesis carried out by pol V, which mostly yields base substitutions at a...