We have determined the DNA sequence changes in 487 spontaneous mutations in the N-terminal part of the lacI gene in mutH, mutL, and mutS strains of Escherichia coli. These strains display elevated spontaneous mutation rates because of a deficiency in the process of postreplicative mismatch correction. As a consequence the mutational spectra reveal the nature of spontaneous DNA replication errors. The spectra consist of base substitutions (75%) and single-base deletions (25%). Among the base substitutions, transitions (both A.T-*G.C and G'C->AT) are strongly favored over transversions (96% versus 4%). Large site-to-site differences are observed among identical base substitutions, presumably reflecting the modulating effects of neighboring bases. The single-base-deletion spectrum is dominated by a large hotspot at a run of adjacent identical base pairs, implying a Streisingerslippage mechanism. The data, when compared to a previously determined wild-type spectrum, also provide information on the specificity of the mismatch repair system. Escherichia coli strains that carry a mutation in the mutH, mutL, or mutS genes display elevated spontaneous mutation rates because they are defective in methylation-instructed DNA mismatch correction (1-8). This system is thought to follow the replication fork, scrutinizing the newly replicated DNA for mismatches resulting from errors of DNA replication. The mismatches are then corrected using the undermethylation (at GATC sites) of the newly synthesized strand to distinguish the correct from the incorrect half of the mismatch.Here, we have made use of the mismatch-repair-deficient strains to further investigate the mechanisms of mutation in E. coli. We previously reported in detail on the DNA sequence changes in a large collection of spontaneous mutants in the lacd gene in a wild-type strain (9). Diverse mutational classes were seen, presumably resulting from different mutational mechanisms. We now report on the DNA sequence changes in 487 mutants in mutH, mutL, and mutS strains. Because errors of DNA replication in these strains are no longer corrected, the data provide us with an intimate view on the nature of in vivo DNA replication errors. The data, in conjunction with the wild-type data, also allow an estimation of the efficiency of the mismatch-repair system for several mutational classes. MATERIALS AND METHODSBacterial Strains. Escherichia coli strains NR3835 (ara, thi, trpE9777, Aprolac, F'prolac), NR3939 (ara, thi, mutH101, Aprolac, F'prolac), NR3940 (ara, thi, trpE9777, mutL101, Aprolac, F'prolac), and NR3996 (ara, thi, trpE9777, mutS-101, Aprolac, F'prolac), all derivatives of strain GM1 (10), were obtained from B. W. Glickman (York University, Toronto). The isolation and characterization of the mutator alleles has been described (2). The F'prolac carries the IQ(IacI) and L8(lacZ) promoter mutations. Strains CSH51, CSH52, and S90C have been described (10, 11).Media. Luria broth (LB) and minimal media were used as described (9). P-gal plates, used for the selec...

To better understand the mechanisms of SOS mutagenesis in the bacterium Escherichia coli, we have undertaken a genetic analysis of the SOS mutator activity. The SOS mutator activity results from constitutive expression of the SOS system in strains carrying a constitutively activated RecA protein (RecA730). We show that the SOS mutator activity is not enhanced in strains containing deficiencies in the uvrABC nucleotide excision-repair system or the xth and nfo base excision-repair systems. Further, recA730-induced errors are shown to be corrected by the MutHLS-dependent mismatch-repair system as efficiently as the corresponding errors in the rec ؉ background. These results suggest that the SOS mutator activity does not reflect mutagenesis at so-called cryptic lesions but instead represents an amplification of normally occurring DNA polymerase errors. Analysis of the base-pair-substitution mutations induced by recA730 in a mismatch repair-deficient background shows that both transition and transversion errors are amplified, although the effect is much larger for transversions than for transitions. Analysis of the mutator effect in various dnaE strains, including dnaE antimutators, as well as in proofreading-deficient dnaQ (mutD) strains suggests that in recA730 strains, two types of replication errors occur in parallel: (i) normal replication errors that are subject to both exonucleolytic proofreading and dnaE antimutator effects and (ii) recA730-specific errors that are not susceptible to either proofreading or dnaE antimutator effects. The combined data are consistent with a model suggesting that in recA730 cells error-prone replication complexes are assembled at sites where DNA polymerization is temporarily stalled, most likely when a normal polymerase insertion error has created a poorly extendable terminal mismatch. The modified complex forces extension of the mismatch largely at the exclusion of proofreading and polymerase dissociation pathways. SOS mutagenesis targeted at replication-blocking DNA lesions likely proceeds in the same manner.One major pathway of mutagenesis in Escherichia coli depends on the SOS response (for a recent review, see reference 28). The SOS response involves the coordinated induction of a set of 20 or more genes upon blockage of ongoing DNA replication. Such blockage can occur by introduction of DNA damage, for example, by UV irradiation or chemical exposure. The SOS system functions to allow the organism to cope with the adverse conditions, to repair or bypass DNA lesions, and to eventually resume replication. During the period of SOS induction, mutations are fixed as well. In the case of UV irradiation, the importance of the SOS system is evidenced by greatly reduced mutagenesis and poor survival if SOS induction is prevented. The term SOS was proposed based on the observed linkage between survival, mutagenesis, and the induction of the response (49, 50, 76). It was thought that SOS induction would allow replicative bypass of some of the blocking lesions, increasing the probabili...

No abstract

We have shown previously that Escherichia coli and Salmonella enterica serovar Typhimurium strains carrying a deletion of the uvrB-bio region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to the uvrB excision repair defect associated with this deletion because a uvrB point mutation or a uvrA deficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in the moeA gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by the uvrB-bio deletion. The moeAB operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as moa or mod, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.The biological effects of many mutagenic agents are due to DNA base modifications, both in the DNA and the DNA precursor pool. A group of mutagens containing a preformed modified base, often referred to as base analogs (14), have received increasing attention recently. For example, 8-oxoguanine, in the form of 8-oxo-dGTP or 8-oxo-GTP, is a spontaneously arising guanine oxidation product that contributes substantially to the infidelity of DNA replication (13,35,37) or transcription (58). Specialized systems protecting the cell against 8-oxoguanine have been found in organisms from bacteria to humans (for reviews, see references 13 and 37), including the MutT enzyme, which is capable of hydrolyzing 8-oxodGTP, an activity referred to as pool sanitizing (2,35). Other examples of mutagenic precursor pool contaminants are 5-hydroxy-dCTP (12) and 2-hydroxy-dATP (15), both oxidative stress products. The human MutT homolog hMTH1 has strong activity towards 2-hydroxy-dATP, suggesting that it, in addition to 8-oxo-GTP, could be an important threat if not actively removed (15). In addition, base analogs can be useful tools for probing the mechanisms of mutation avoidance during DNA replication, including base-base discrimination by DNA polymerases (51, 54).An important group of base analogs are the N-hydroxy derivatives of adenine and cytidine (see reference 27 for a review). For example, 6-hydroxylaminopurine (N-6-hydroxyadenine) (HAP) and 2-amino-6-hydroxylaminopurine (AHAP) are very powerful mutagens in phage, bacteria, yeast, and eukaryotic cells (42, 43), and they have been termed universal mutagens (42). These adenine derivatives are active when provided as bases or, in some organisms, nucleosides, as they are apparently converted efficiently into the corresponding deoxynucleoside triphosphates (dNTPs), which are then incorporated into DNA by DNA po...

Mutation induced by ultraviolet light is predominantly targeted by UV photoproducts. Two primary candidates for the premutagenic lesion are the cyclobutane pyrimidine dimer and the less frequent (by a factor of 10) pyrimidine-pyrimidone (6-4) photoproduct. Methylation of the 3'-cytosine in the sequence 5' CCAGG 3' reduces the yield of (6-4) lesions, but not of cyclobutane dimers, at these sites. By taking advantage of mutants deficient in cytosine methylation, we show here that at the three sites in the lacI gene of Escherichia coli having this sequence, the specific increase in the formation of the (6-4) photoproducts is accompanied by a concomitant increase in mutation. At each site, a GC to APT transition results in an amber mutation. In the unmethylated state, these sites become among the most frequent nonsense mutations recovered. We conclude that the (6-4) photoproduct constitutes a major premutagenic lesion in E. coli.