DNA Polymerase III from Escherichia coliCells Expressing mutA Mistranslator tRNA Is Error-prone

Abstract

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...

Culture History and Population Heterogeneity as Determinants of Bacterial Adaptation: the Adaptomics of a Single Environmental Transition

“…However, phenotypic diversity generated by mistranslation can potentially allow heritable adaptation. The discovery in E. coli and other bacteria of a hypermutagenesis phenotype associated with codon ambiguity, the "translational stress mutagenesis" (TSM) phenotype, provides a potential solution to the problem of heritable transmission of protein translation errors (1)(2)(3)77). Ambiguous codon decoding expands the proteome and generates new phenotypes.…”

Escherichia coli Cells Bearing a Ribosomal Ambiguity Mutation in rpsD Have a Mutator Phenotype That Correlates with Increased Mistranslation

“…Interestingly, the mutator phenotype induced by mutA is suppressed in cells defective for RecABC-mediated homologous recombination functions (20,21). mutA cells have an errorprone DNA polymerase activity (2) that is most likely to be a modified form of DNA polymerase III (1). The mechanism by which mistranslation leads to the TSM phenotype is not known, but two hypotheses are considered elsewhere in this communication.…”

“…The induced pathway hypothesis generalizes the mechanism to many types of mistranslation beyond Asp3Gly misreading and postulates that increased translational errors generate a signal (increased protein turnover or creation of gain-of-function mutant proteins) that induces a pathway that ultimately modifies DNA polymerase III (1,2,6,10,12,20,21). Thus, both hypotheses invoke a modification of DNA polymerase III, a prediction that has recently been verified (1,22). However, the two hypotheses differ in the proposed mechanisms for the modification, and experimental evidence available to date cannot be unequivocally interpreted as supporting one mechanism over the other.…”

An editing-defective aminoacyl-tRNA synthetase is mutagenic in aging bacteria via the SOS response

“…3). Although work by others (29)(30)(31) showed translational errors could increase mutation rates under certain conditions, the connection to the SOS response established in this work may be specific to the way that mistranslation was induced. Because of functional ablation of the editing domain of the isoleucyl-tRNA synthetase, all proteins are synthesized as statistical entities, meaning that isoleucine is replaced at a low frequency by valine (and other naturally occurring loose structural analogs of isoleucine).…”

“…These mutants replace the tRNA anticodon for glycine with that for asparagine, resulting in statistical proteins that incur Asp 3 Gly substitutions. The source of the increased mutation rate is DnaQ, which provides an error-correcting exonuclease function to the replicative DNA pol III (30). This cause of increased mutation rate is known as translational stress-induced mutagenesis (TSM).…”