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Cells lacking deoxycytidine deaminase (DCD) have been shown to have imbalances in the normal dNTP pools that lead to multiple phenotypes, including increased mutagenesis, increased sensitivity to oxidizing agents, and to a number of antibiotics. In particular, there is an increased dCTP pool, often accompanied by a decreased dTTP pool. In the work presented here, we show that double mutants of Escherichia coli lacking both DCD and NDK (nucleoside diphosphate kinase) have even more extreme imbalances of dNTPs than mutants lacking only one or the other of these enzymes. In particular, the dCTP pool rises to very high levels, exceeding even the cellular ATP level by several-fold. This increased level of dCTP, coupled with more modest changes in other dNTPs, results in exceptionally high mutation levels. The high mutation levels are attenuated by the addition of thymidine. The results corroborate the critical importance of controlling DNA precursor levels for promoting genome stability. We also show that the addition of certain exogenous nucleosides can influence replication errors in DCD-proficient strains that are deficient in mismatch repair.

We have previously described a novel Escherichia coli detoxification system for the removal of toxic and mutagenic N-hydroxylated nucleobases and related compounds that requires the molybdenum cofactor. Two subpathways (ycbX and yiiM) were identified, each employing a novel molybdo activity capable of inactivating N-hydroxylated compounds by reduction to the corresponding amine. In the present study, we identify the cysJ gene product as one additional component of this system. While the CysJ protein has been identified as the NADPH:flavin oxidoreductase component of the CysJI sulfite reductase complex (Cys[J.sub.8][I.sub.4]), we show that the role of CysJ in base analog detoxification is unique and independent of CysI and sulfite reductase. We further show that CysJ functions as a specific partner of the YcbX molybdoenzyme. We postulate that the function of CysJ in this pathway is to provide, via its NADPH:flavin reductase activity, the reducing equivalents needed for the detoxification reaction at the YcbX molybdocenter. In support of the proposed interaction of the CysJ and YcbX proteins, we show that an apparent CysJ-YcbX 'hybrid' protein from two Vibrio species is capable of compensating for a double cysJ ycbX defect in E. coli. doi: 10.1128/JB.01438-09

A novel mutator locus in Escherichia coli was identified from a collection of random transposon insertion mutants. Several mutators in this collection were found to have an insertion in the dgt gene, encoding a previously characterized dGTP triphosphohydrolase. The mutator activity of the dgt mutants displays an unusual specificity. Among the six possible base pair substitutions in a lacZ reversion system, the G * C[right arrow]C * G transversion and A * T [right arrow] G * C transition are strongly enhanced (10- to 50-fold), while a modest effect (two- to threefold) is also observed for the G * C [right arrow] A * T transition. Interestingly, a two- to threefold reduction in mutant frequency (antimutator effect) is observed for the G * C [right arrow] T * A transversion. In the absence of DNA mismatch repair (mutL) some of these effects are reduced or abolished, while other effects remain unchanged. Analysis of these effects, combined with the DNA sequence contexts in which the reversions take place, suggests that alterations of the dGTP pools as well as alterations in the level of some modified dNTP derivatives could affect the fidelity of in vivo DNA replication and, hence, account for the overall mutator effects.

The dnaX36(TS) mutant of Escherichia coli confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (-1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various E. coli DNA polymerases. The dnaX gene encodes the x subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The dnaX36 defect resides in the C-terminal domain V of [tau], essential for interaction of x with the a (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered [alpha]-[tau] interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of dnaX36 with mutM, mutY, and recA defects, support our assumption that dnaX36-mediated mutations originate as errors of replication rather than DNA damagerelated events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (dinB) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of [tau] subunit, in securing a high fidelity of replication.

Constitutive expression of the SOS regulon in Escherichia coli recA730 strains leads to a mutator phenotype (SOS mutator) that is dependent on DNA polymerase V (umuDC gene product). Here we show that a significant fraction of this effect also requires DNA polymerase IV (dinB gene product).

The Hot (homolog of theta) protein of bacteriophage P1 can substitute for the Escherichia coli DNA polymerase III [theta] subunit, as evidenced by its stabilizing effect on certain dnaQ mutants that carry an unstable polymerase III [epsilon] proofreading subunit (antimutator effect). Here, we show that Hot can also cause an increase in the mutability of various E. coli strains (mutator effect). The hot mutator effect differs from the one caused by the lack of [theta]. Experiments using chimeric [theta]/Hot proteins containing various domains of Hot and [theta] along with a series of point mutants show that both N- and C-terminal parts of each protein are important for stabilizing the [epsilon] subunit. In contrast, the N-terminal part of Hot appears uniquely responsible for its mutator activity.

The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: [alpha], [epsilon], and [theta]. The [alpha] subunit contains the polymerase, and the [epsilon] subunit contains the exonucleolytic proofreading function. The small (8-kDa) [theta] subunit binds only to [epsilon]. Its function is not well understood, although it was shown to exert a small stabilizing effect on the [epsilon] proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, [theta] yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the [theta] homolog from bacteriophage P1, named HOT, we constructed a homology model of [theta]. This model suggested that the unfavorable behavior of [theta] might arise from exposed hydrophobic residues, particularly toward the end of [alpha]-helix 3. In gel filtration studies, [theta] elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded [sup.1]H-[sup.15]N heteronuclear single quantum correlation (HSQC) spectra in water-alcohol mixed solvents and observed substantially improved dispersion and uniformity of peak intensities, facilitating a structural determination under these conditions. The structure of [theta] in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported [theta] structure. The new [theta] structure is expected to provide additional insight into its physiological role and its effect on the [epsilon] proofreading subunit.

We investigated the mutator effect resulting from overproduction of Escherichia coli DNA polymerase IV. Using lac mutational targets in the two possible orientations on the chromosome, we observed preferential mutagenesis during lagging strand synthesis. The mutator activity likely results from extension of mismatches produced by polymerase III holoenzyme.

The [theta] subunit (holE gene product) of Escherichia coli DNA polymerase (Pol) III holoenzyme is a tightly bound component of the polymerase core. Within the core ([alpha]-[epsilon]-[theta]), the [alpha] and [epsilon] subunits carry the DNA polymerase and 3' proofreading functions, respectively, while the precise function of [theta] is unclear, holE homologs are present in genomes of other enterobacteriae, suggestive of a conserved function. Putative homologs have also been found in the genomes of bacteriophage P1 and of certain conjugative plasmids. The presence of these homologs is of interest, because these genomes are fully dependent on the host replication machinery and contribute few, if any, replication factors themselves. To study the role of these [theta] homologs, we have constructed an E. coli strain in which holE is replaced by the P1 homolog, hot. We show that hot is capable of substituting for holE when it is assayed for its antimutagenic action on the proofreading-impaired dnaQ49 mutator, which carries a temperature-sensitive [epsilon] subunit. The ability of hot to substitute for holE was also observed with other, although not all, dnaQ mutator alleles tested. The data suggest that the P1 hot gene product can substitute for the [theta] subunit and is likely incorporated in the Pol III complex. We also show that overexpression of either [theta] or Hot further suppresses the dnaQ49 mutator phenotype. This suggests that the complexing of dnaQ49-[epsilon] with [theta] is rate limiting for its ability to proofread DNA replication errors. The possible role of hot for bacteriophage P1 is discussed.

We have investigated whether DNA polymerase IV (Pal IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli. We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome. Virtually no contribution of Pol IV to the chromosomal mutation rate was observed. On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F'(pro-lac) episome.

The function of the [theta] subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. [theta] is a tightly bound component of the DNA polymerase III core, which contains the a subunit (polymerase), the [epsilon] subunit (3' [right arrow] 5' exonuclease), and the [theta] subunit, in the linear order [alpha]-[epsilon]-[theta]. Previous studies have shown that the [theta] subunit is not essential, as strains carrying a deletion of the holE gene (which encodes [theta]) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3'-exonuclease activity of [epsilon] to be modestly enhanced by the presence of [theta]. Here, we report a series of genetic experiments that suggest that [theta] has a stabilizing role for the [epsilon] proofreading subunit. The observations include (i) defined [DELTA]holE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong [DELTA]holE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of [alpha]-[epsilon] interactions by the presence of [theta]. [theta] appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of [theta] might be uniquely beneficial in those instances where the proofreading 3'-exonuclease is not part of the polymerase polypeptide.

Deoxyribosyl-dihydropyrimido [4,5-c] [1,2] oxazin-7-one (dP) is a potent mutagenic deoxycytidine-derived base analogue capable of pairing with both A and G, thereby causing G * C [right arrow] A * T and A * T [right arrow] G * C transition mutations. We have found that the Escherichia coli DNA mismatch-repair system can protect cells against this mutagenic action. At a low dose, dP is much more mutagenic in mismatch-repair-defective mutH, mutL, and mutS strains than in a wild-type strain. At higher doses, the difference between the wild-type and the mutator strains becomes small, indicative of saturation of mismatch repair. Introduction of a plasmid containing the E. coli mut[L.sup.+] gene significantly reduces dP-induced mutagenesis. Together, the results indicate that the mismatch-repair system can remove dP-induced replication errors, but that its capacity to remove dP-containing mismatches can readily be saturated. When cells are cultured at high dP concentration, mutant frequencies reach exceptionally high levels and viable cell counts are reduced. The observations are consistent with a hypothesis in which dP-induced cell killing and growth impairment result from excess mutations (error catastrophe), as previously observed spontaneously in proofreading-deficient mutD (dnaQ) strains.

The frequency of UV-induced mutations in excision-repair-deficient strains of Escherichia coli is measured by determining UV-induced reversions of six different lacZ alleles. Data do not suggest that SOS-mediated mutagenic translesion synthesis probably occurs with equal or similar orientations on leading and lagging strands.

Research has been conducted on the DNA polymerase III holoenzyme, the replicative Escherichia coli polymerase. The development of the (super)2 H-, (super)13 C- and (super)15 N-labeled N-terminal domain (epsilon186) has been carried out and the results indicate that domain is structurally similar to the other evolutionary distant proofreading exonucleases.

Research has been conducted on the deleted uvrB-bio region in Escherichia coli and Salmonella enterica serovar Typhimurium strains. Genes responsible for this deletion have been investigated.

Antimutators are mutant strains that have reduced mutation rates compared to the corresponding wild-type strain. Their existence, along with mutator mutants that have higher mutation rates compared to the wild-type strain, are powerful evidence that mutation rates are genetically controlled. Compared to mutator mutants, antimutators have a very distinguishing property. Because they prevent normally occurring mutations, they, uniquely, are capable of providing insight into the mechanisms of spontaneous mutations. In this review, antimutator mutants are discussed in bacteriophage T4 and the bacterium Escherichia coli, with regard to their properties, possible mechanisms, and implications for the sources of spontaneous mutations in these two organisms.

Research was conducted to examine the in vivo protein interactions within the Escherichia coli DNA polymerase III core. The Saccharomyces cerevisiae two-hybrid system was used to analyze specific in vivo protein interactions. Interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE proteins were demonstrated. Results show that the mutator phenotype of dnaQ49 may be the result of a changed conformation of the epsilon protein, which leads to altered interactions within the Pol III core.

Research was conducted to examine the mechanisms of SOS mutagenesis in the bacterium Escherichia coli using a genetic analysis of the SOS mutator activity. Mutant frequencies were evaluated by toothpicking single colonies for each strains while occasional jackpot cultures were removed from the analysis. Results indicated that modification of the replication machinery is not permanent in recA730 cells. They also showed a strong interaction between the dnaE486 and recA730 mutator activities.

The Escherichia coli dnaQ gene encodes the proofreading 3[prime] exonuclease ([Epsilon] subunit) of DNA polymerase III holoenzyme and is a critical determinant of chromosomal replication fidelity. We constructed by site-specific mutagenesis a mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12[approaches]Ala and Glu-14[approaches]Ala) in the Exo I motif, which, by analogy to other proofreading exonucleases, is essential for the catalytic activity. When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, still binds to the polymerase subunit ([Alpha] subunit or dnaE gene product). When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells became inviable. However, viable dnaQ926 strains could be obtained if they contained one of the dnaE alleles previously characterized in our laboratory as antimutator alleles or if it carried a multicopy plasmid containing the E. coli mutL+ gene. These results suggest that loss of proofreading exonuclease activity in dnaQ926 is lethal due to excessive error rates (error catastrophe). Error catastrophe results from both the loss of proofreading and the subsequent saturation of DNA mismatch repair. The probability of lethality by excessive mutation is supported by calculations estimating the number of inactivating mutations in essential genes per chromosome replication.

The dnaE alleles in Escherichia coli are responsible for a significant antimutator effect, and this effects appears to be independent of exonucleolytic proofreading. In a mut5 mutator strain that is deficient in proofreading, dnaE alleles had a significant antimutator effect, probably by restoring DNA mismatch repair. A particularly effective, new dnaE allele was isolated. Possible mechanisms of the dnaE antimutator effect is described.

DNA sequencing and colony hybridization studies of the function of the dnaE911 DNA polymerase antimutator allele in spontaneous mutagenesis of Escherichia coli reveal that dnaE911 reduces galK, lacI and Rif mutations by 1.5 to 2-fold in the mismatch-repair proficient background. DNA sequences of the lacI gene mutants show that transitions, deletions, frameshifts and duplications remain stable, while a 3-fold reduction of transversions occurs in the dnaE911 allele presence. The A.T to T.A transversion undergoes a 6-fold reduction while the A.T to G.C base-pair substitution hotspot in the lac operator undergoes 5-fold decrease, suggesting that dnaE911 allelic activity is genetic background and mutation type specific.

The mutations in approximately 2400 4-aminobiphenyl (4-AB) + S9-induced revertants of the -1 frameshift allele hisD3052 and the base-substitution allele hisG46 of Salmonella typhimurium were analyzed using colony probe hybridization and polymerase chain reaction. Around 85% of the revertants had the hotspot deletion, while around 15% had complex frameshifts. This was evident in strain TA98 (DeltauvrB, pKM101) which bore the SOS DNA repair system of the pKM101 plasmid. These mutations are cryptic complex frameshifts.