Elevated expression of DNA polymerase II increases spontaneous mutagenesis in Escherichia coli

Escherichia coli DNA polymerase II (Pol-II), encoded by the SOS-regulated polB gene, belongs to the highly conserved group B (-like) family of “high-fidelity” DNA polymerases. Elevated expression of polB gene was recently shown to result in a significant elevation of translesion DNA synthesis at 3, N⁴-ethenocytosine lesion with concomitant increase in mutagenesis. Here, I show that elevated expression of Pol-II leads to an approximately 100-fold increase in spontaneous mutagenesis in a manner that is independent of SOS, umuDC, dinB, recA, uvrA and mutS functions. Cells grow slowly and filament with elevated expression of Pol-II. Introduction of carboxy terminus (“β interaction domain”) mutations in polB eliminates elevated spontaneous mutagenesis, as well as defects in cell growth and morphology, suggesting that these abilities require the interaction of Pol-II with the β processivity subunit of DNA polymerase III. Introduction of a mutation in the proofreading exo motif of polB elevates mutagenesis by a further 180-fold, suggesting that Pol-II can effectively compete with DNA polymerase III for DNA synthesis. Thus, Pol-II can contribute to spontaneous mutagenesis when its expression is elevated.     

Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme

Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution. The resulting DNA was transfected into E. coli JM101 without further treatment. Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum. Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis. Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.     

Escherichia coli DNA polymerase IV contributes to spontaneous mutagenesis at coding sequences but not microsatellite alleles

Slipped strand mispairing during DNA synthesis is one proposed mechanism for microsatellite or short tandem repeat (STR) mutation. However, the DNA polymerase(s) responsible for STR mutagenesis have not been determined. In this study, we investigated the effect of the Escherichia colidinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability, using an HSV-tk gene episomal reporter system for microsatellite mutations. For the control vector (HSV-tk gene only) we observed a statistically significant 3.5-fold lower median mutation frequency in dinB(-) than dinB(+) cells (p<0.001, Wilcoxon Mann Whitney Test). For vectors containing an in-frame mononucleotide allele ([G/C](10)) or either of two dinucleotide alleles ([GT/CA](10) and [TC/AG](11)) we observed no statistically significant difference in the overall HSV-tk mutation frequency observed between dinB(+) and dinB(-) strains. To determine if a mutational bias exists for mutations made by Pol IV, mutational spectra were generated for each STR vector and strain. No statistically significant differences between strains were observed for either the proportion of mutational events at the STR or STR specificity among the three vectors. However, the specificity of mutational events at the STR alleles in each strain varied in a statistically significant manner as a consequence of microsatellite sequence. Our results indicate that while Pol IV contributes to spontaneous mutations within the HSV-tk coding sequence, Pol IV does not play a significant role in spontaneous mutagenesis at [G/C](10), [GT/CA](10), or [TC/AG](11) microsatellite alleles. Our data demonstrate that in a wild type genetic background, the major factor influencing microsatellite mutagenesis is the allelic sequence composition.     

Mutagenic DNA repair in Escherichia coli. XIV. Influence of two DNA polymerase III mutator alleles on spontaneous and UV mutagenesis

Summary We introduced the dnaE486 and polC74 mutations (which are associated with decreased DNA polymerase III replication fidelity) into excision defective Escherichia coli strains with varying SOS responses. These mutations increased the UV-induced frequency of base pair substitution mutations in all strains tested, except recA430 and umuC122 derivatives. This UV mutator effect therefore requires expression of the SOS error-prone repair system. In recA441 lexA51 strains where the SOS system is constitutively expressed, the UV mutator effect of the dnaE alleles was similar in relative terms (though greater in absolute terms). Since these dnaE alleles decrease rather than increase survival after UV it is argued that they promote a burst of untargeted mutations close to UV photoproducts (hitch-hiking mutations) rather than increase the number of translesion synthesis events. The fact that there was no UV mutagenesis in dnaE486 umuC122 or polC74 umuC122 strains indicates that infidelity associated with these dnaE alleles did not of itself enable translesion synthesis to occur. The spontaneous mutator effect conferred by dnaE486 and polC74 was not affected by umuC122 or recA430 indicating that it is not dependent upon error-prone repair ability. In recA441 lexA51 bacteria, where SOS error-prone repair is constitutively induced, the mutator effect of dnaE486 was greater and was largely blocked by umuC122. It is suggested that spontaneously occurring cryptic lesions that are themselves unable to induce the SOS system are subject to translesion synthesis under these conditions and trigger a burst of hitch-hiking mutations that are therefore effectively umuC dependent.     

Expression of the Fpg protein of Escherichia coli in Saccharomyces cerevisiae: effects on spontaneous mutagenesis and sensitivity to oxidative DNA damage

The biological relevance of oxidative DNA damage has been unveiled by the identification of genes such as fpg of E. coli or OGG1 of Saccharomyces cerevisiae. Both Fpg and Ogg1 proteins are DNA glycosylases/AP lyases that excise 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyG) from damaged DNA. Although similar, the enzymatic and biological properties of Fpg and Ogg1 proteins are not identical. Furthermore, the Fpg and Ogg1 proteins do not show significant sequence homologies. In this study, we investigated the ability of the Fpg protein of E. coli to complement phenotypes thought to be due to oxidative DNA damage in Saccharomyces cerevisiae. To express Fpg in yeast, the coding sequence of the fpg gene was placed under the control of a strong yeast promoter in the expression vector pCM190 to generate the pFPG240 plasmid. The Ogg1-deficient yeast strain CD138, ogg1::TRP1, was transformed with pFPG240 and the expression of Fpg was measured. Expression of Fpg in yeast harboring pFPG240 was revealed by efficient release of Me-FapyG and cleavage of 8-OxoG-containing duplexes by cell free protein extracts. The production of the Fpg protein in yeast cells was further demonstrated by immunoblotting analysis using anti-Fpg antibodies. Fpg expression suppresses the spontaneous mutator phenotype of ogg1- yeast for the production of canavanin resistant mutants (CanR) and Lys+ revertants. Fpg expression also restores the capacity of plasmid DNA treated with methylene blue plus visible light (MB-light) to transform the yeast ogg1- rad1- double mutant.     

Mutator phenotype resulting from DNA polymerase IV overproduction in Escherichia coli: preferential mutagenesis on the lagging strand

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.     

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.     

The SMC-like protein complex SbcCD enhances DNA polymerase IV-dependent spontaneous mutation in Escherichia coli

In Escherichia coli, RpoS, the general stress response sigma factor, regulates the activity of the specialized DNA polymerase DNA polymerase IV (Pol IV) both in stationary-phase and in exponential-phase cells. Because during exponential phase dinB, the gene encoding Pol IV, is transcribed independently of RpoS, RpoS must regulate Pol IV activity in growing cells indirectly via one or more intermediate factors. The results presented here show that one of these intermediate factors is SbcCD, an SMC-like protein and an ATP-dependent nuclease. By initiating or participating in double-strand break repair, SbcCD may provide DNA substrates for Pol IV polymerase activity.     

Monofunctional alkylating agent-induced inactivation, mutagenesis and DNA degradation in an Escherichia coli mutant deficient in DNA polymerase

Summary The DNA polymerase deficient mutantE. coli P3478polA1 is extremely sensitive to the lethal action of N-methyl-N-nitro-N-nitrosoguanidine (NG) and methyl-methanesulfonate (MMS). ThepolA1 mutant has an almost unaffected mutability induced by NG or MMS and shows reduced ability to propagate MMS-treated phage T7. NG and MMS induce marked breakdown of DNA and inhibit significantly DNA synthesis in thepolA1 mutant. The obtained results suggest the thepolA1 mutant is unable to repair single-strand breaks of DNA induced by monofunctional alkylating agents. The suggestion is confirmed by the demonstrated sensitivity of thepolA1 mutant to thymine starvation (TS).     

DNA sequence context affects UV-induced mutagenesis in Escherichia coli

We investigated the effect of altering the DNA sequence surrounding a mutable target site on the production of ultraviolet light (UV) induced mutations. Site-directed base substitutions were incorporated on both sides of a TAA sequence encoding a UAA nonsense defect in the tyrA14 allele of Escherichia coli. This allele is readily revertable by UV and a total of eight different base substitution mutations can be recovered. Five different strains harboring DNA sequences allowing the formation of 5'-TT, 5'-CT and 5'-TA* photoproducts were constructed and exposed to UV. DNA sequence analysis was used to determine the spectrum of the revertants that were recovered. The results showed that changes at the 3'-base of a TT site were predominantly T to C transitions and T to A transversions. However, unlike the TT site, a 5'-CT site produced a relatively high frequency of T to G transversions. In addition, T to A transversions that could not have been targeted by a cyclobutane-type or [6-4]-type pyrimidine dimer were produced; this result suggested that these mutations may be targeted by a TA* photoproduct. Also, a distinct strand bias was noted for two mechanistically identical base substitutions in a strain having a palindromic target sequence; this result may reflect an unequal damage distribution or processing of photoproducts as a consequence of asymmetric DNA replication. Finally, our results show that DNA sequences expected to allow the greatest density of UV-induced DNA damage produce the highest mutation frequencies. Overall, these findings provide new insights regarding the role of DNA photoproducts in UV mutagenesis.     

Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli

Antimutagenic effects of cinnamaldehyde on mutagenesis by chemical agents were investigated in Escherichia coli WP2 uvrA- trpE-. Cinnamaldehyde, when added to agar medium, greatly reduced the number of Trp+ revertants induced by 4-nitroquinoline 1-oxide (4-NQO) without any decrease of cell viability. This antimutagenic effect could not be explained by inactivation of 4-NQO caused by direct interaction with cinnamaldehyde. Mutagenesis by furylfuramide (AF-2) was also suppressed significantly. Mutations induced by methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) were slightly inhibited. However, cinnamaldehyde was not at all effective on the mutagenesis of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Two derivatives of cinnamaldehyde, cinnamyl alcohol and trans-cinnamic acid, did not have as strong antimutagenic effects on 4-NQO mutagenesis as cinnamaldehyde had. Because cinnamaldehyde showed marked antimutagenic effects against mutations induced by UV-mimic mutagens but not those induced by MNNG or EMS, it seems that cinnamaldehyde might act by interfering with an inducible error-prone DNA repair pathway.     

Expression of mutant alanine tRNAs increases spontaneous mutagenesis in Escherichia coli

The expression of mutA, an allele of the glycine tRNA gene glyV, can confer a novel mutator phenotype that correlates with its ability to promote Asp-->Gly mistranslation. Both activities are mediated by a single base change within the anticodon such that the mutant tRNA can decode aspartate codons (GAC/U) instead of the normal glycine codons (GCC/U). Here, we investigate whether specific Asp-->Gly mistranslation is required for the unexpected mutator phenotype. To address this question, we created and expressed 18 individual alleles of alaV, the gene encoding an alanine tRNA, in which the alanine anticodon was replaced with those specifying other amino acids such that the mutant (alaVX) tRNAs are expected to potentiate X-->Ala mistranslation, where X is one of the other amino acids. Almost all alaVX alleles proved to be mutators in an assay that measured the frequency of rifampicin-resistant mutants, with one allele (alaVGlu) being a stronger mutator than mutA. The alaVGlu mutator phenotype resembles that of mutA in mutational specificity (predominantly transversions), as well as SOS independence, but in a puzzling twist differs from mutA in that it does not require a functional recA gene. Our results suggest that general mistranslation (as opposed to Asp-->Gly alone) can induce a mutator phenotype. Furthermore, these findings predict that a large number of conditions that increase translational errors, such as genetic defects in the translational apparatus, as well as environmental and physiological stimuli (such as amino acid starvation or exposure to antibiotics) are likely to activate a mutator response. Thus, both genetic and epigenetic mechanisms can accelerate the acquisition of mutations.     

Overproduction of the beta subunit of DNA polymerase III holoenzyme reduces UV mutagenesis in Escherichia coli.

Overproduction of the beta subunit of DNA polymerase III holoenzyme caused a 5- to 10-fold reduction of UV mutagenesis along with a slight increase in sensitivity to UV light in Escherichia coli. The same effects were observed in excision-deficient cells, excluding the possibility that they were mediated via changes in excision repair. In contrast, overproduction of the alpha subunit of the polymerase did not influence either UV mutagenesis or UV sensitivity. The presence of the mutagenesis proteins MucA and MucB expressed from a plasmid alleviated the effect of overproduced beta on UV mutagenesis. We have previously suggested that DNA polymerase III holoenzyme can exist in two forms: beta-rich form unable to bypass UV lesions and a beta-poor form capable of bypassing UV lesions (O. Shavitt and Z. Livneh, J. Biol. Chem. 264:11275-11281, 1989). The beta-poor form may be related to an SOS form of DNA polymerase III designed to perform translesion polymerization under SOS conditions and thereby generate mutations. On the basis of this model, we propose that the overproduced beta subunit affects the relative abundance of the regular replicative beta-rich polymerase and the SOS bypass-proficient polymerase by sequestering the polymerase molecules to the beta-rich form and blocking the SOS form.     

Frameshift mutagenesis in Escherichia coli by reversible DNA intercalators: sequence specificity

The mutagenic potency of the simple reversible intercalators isopropyl-OPC (iPr-OPC) and 9-aminoacridine (9-AA) is assessed in E. coli using reversion assays based on plasmids derived from pBR322 carrying various frameshift mutations within the tetracycline resistance gene in repetitive sequences: +/- 2 frameshift mutations within alternating GC sequences; +/- 1 frameshift mutation at runs of guanines. The results obtained show that iPr-OPC and 9-AA have a sequence specificity for mutagenesis: they revert +1 and -1 frameshift mutations within runs of monotonous G:C base pairs. The precise determination of the size of a small restriction fragment which contains the mutation allowed us to demonstrate that reversion occurred by -1 deletions for the +1 frameshift mutations and by +1 additions for the -1 frameshift mutations. The possible relations of this specific reversion with the base sequence specificity of the mutagenesis are briefly discussed.     

Mutagenic DNA repair in Escherichia coli XIII Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis

We have introduced a mutD5 mutation (which results in defective 3′–5′-exonuclease activity of the ϵ proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light. MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives. It had no effect of UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria. It is concluded that the ϵ proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.     

Molecular actions of Escherichia coli MutT for control of spontaneous mutagenesis

MutT protein of Escherichia coli hydrolyzes oxidized guanine nucleotides, 8-oxo-dGTP and 8-oxoGTP, to the corresponding monophosphates, thereby preventing misincorporation of 8-oxoguanine into DNA and RNA, respectively. Although the biological significance of the MutT has been established, how MutT protein actually works in vivo remains to be elucidated. The current study shows the molecular behavior of the MutT protein in vivo and in vitro with special reference to control of spontaneous mutagenesis. A single E. coli cell carries about 70-75 molecules of the MutT protein and that this number does not change even when the cells were cultured in anaerobic and hyper-oxidative conditions. Conditional gene silencing analyses revealed that about a half number of MutT molecules are needed for keeping the spontaneous mutation frequency at the normal level. The MutT functions are not needed under anaerobic condition, yet the level of the MutT protein in cell is kept constant, probably for preparing for sudden changes of oxygen pressure. There is a possibility that MutT functions in close association with other proteins, and evidence is presented that MutT protein can interact with some proteins in vivo.