AlkB dioxygenase in preventing MMS-induced mutagenesis in Escherichia coli: Effect of Pol V and AlkA proteins (original) (raw)
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Mutation Research/ …, 2010
In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense 23 systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have 24 previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells 25 from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), α-26 ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in 27 DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA 28 repair (TCR) on MMS-induced mutagenesis in E.coli strain deficient in functional AlkB protein. 29 Measuring the decline in the frequency of MMS-induced argE3→Arg + revertants under transient 30 amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the 31 BS87 (alkB -) strain in comparison with the AB1157 (alkB + ) counterpart. Mutation in the mfd 32 gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-33 treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd + , and no repair in BS87 34 mfdcells. Determination of specificity of Arg + revertants allowed to conclude that MMS-35 induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of 36 mutations. These include AT→TA transversions by supL suppressor formation (1meA) and 37 GC→AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly 38 Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The 39 nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-40 terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in 41 the alkBmfdbacteria but not in the alkB + mfdcounterpart. 42 43 44
Mutagenesis, 2009
Methylmethane sulphonate (MMS), an S N 2-type alkylating agent, generates DNA methylated bases exhibiting cytotoxic and mutagenic properties. Such damaged bases can be removed by a system of base excision repair (BER) and by oxidative DNA demethylation catalysed by AlkB protein. Here, we have shown that the lack of the BER system and functional AlkB dioxygenase results in (i) increased sensitivity to MMS, (ii) elevated level of spontaneous and MMS-induced mutations (measured by argE3 / Arg 1 reversion) and (iii) induction of the SOS response shown by visualization of filamentous growth of bacteria. In the xth nth nfo strain additionally mutated in alkB gene, all these effects were extreme and led to 'error catastrophe', resulting from the presence of unrepaired apurinic/apyrimidinic (AP) sites and 1-methyladenine (1meA)/3-methylcytosine (3meC) lesions caused by deficiency in, respectively, BER and AlkB dioxygenase. The decreased level of MMS-induced Arg 1 revertants in the strains deficient in polymerase V (PolV) (bearing the deletion of the umuDC operon), and the increased frequency of these revertants in bacteria overproducing PolV (harbouring the pRW134 plasmid) indicate the involvement of PolV in the error-prone repair of 1meA/3meC and AP sites. Comparison of the sensitivity to MMS and the induction of Arg 1 revertants in the double nfo alkB and xth alkB, and the quadruple xth nth nfo alkB mutants showed that the more AP sites there are in DNA, the stronger the effect of the lack of AlkB protein. Since the sum of MMS-induced Arg 1 revertants in xth, nfo and nth xth nfo and alkB mutants is smaller than the frequency of these revertants in the BER 2 alkB 2 strain, we consider two possibilities: (i) the presence of AP sites in DNA results in relaxation of its structure that facilitates methylation and (ii) additional AP sites are formed in the BER 2 alkB 2 mutants.
Mutagenic potency of MMS‐induced 1meA/3meC lesions in E. coli
Environmental and …, 2009
The mutagenic activity of MMS in E. coli depends on the susceptibility of DNA bases to methylation and their repair by cellular defense systems. Among the lesions in methylated DNA is 1meA/ 3meC, which is recently recognized as being mutagenic. In this report, special attention is focused on the mutagenic properties of 1meA/3meC which, by the activity of AlkB-dioxygenase, are quickly and efficiently converted to natural A/C bases in the DNA of E. coli alkB 1 strains, preventing 1meA/ 3meC-induced mutations. We have found that in the absence of AlkB-mediated repair, MMS treatment results in an increased frequency of four types of base substitutions: GC?CG, GC?TA, AT?CG, and AT?TA, whereas overproduction of PolV in CC101-106 alkB 2 /pRW134 strains leads to a markedly elevated level of GC?TA, GC?CG, and AT?TA transversions. It has been observed V C 2009 Wiley-Liss, Inc.
The Escherichia coli alkA Gene Is Activated to Alleviate Mutagenesis by an Oxidized Deoxynucleoside
Frontiers in Microbiology, 2020
The cellular methyl donor S-adenosylmethionine (SAM) and other endo/exogenous agents methylate DNA bases non-enzymatically into products interfering with replication and transcription. An important product is 3-methyladenine (m 3 A), which in Escherichia coli is removed by m 3 A-DNA glycosylase I (Tag) and II (AlkA). The tag gene is constitutively expressed, while alkA is induced by sub-lethal concentrations of methylating agents. We previously found that AlkA exhibits activity for the reactive oxygen-induced thymine (T) lesion 5-formyluracil (fU) in vitro. Here, we provide evidence for AlkA involvement in the repair of oxidized bases by showing that the adenine (A) • T → guanine (G) • cytosine (C) mutation rate increased 10-fold in E. coli wild-type and alkA − cells exposed to 0.1 mM 5-formyl-2-deoxyuridine (fdU) compared to a wild-type specific reduction of the mutation rate at 0.2 mM fdU, which correlated with alkA gene induction. G • C → A • T alleviation occurred without alkA induction (at 0.1 mM fdU), correlating with a much higher AlkA efficiency for fU opposite to G than for that to A. The common keto form of fU is the AlkA substrate. Mispairing with G by ionized fU is favored by its exclusion from the AlkA active site.
Mutation research, 1979
Arg+ revertants of E. coli AB1157 and derivative strains were selected after MMS mutagenesis and subjected to a phenotypic analysis which permitted the partitioning of revertants into 4 classes. The distribution of these revertant classes was influenced by mutations affecting DNA-repair systems, mutagen treatment and revertant-selection methods. Introduction of the R46 plasmid into strains also affected this mutational specificity, and it was concluded that the plasmid's mutagenic enhancing effect does not merely augment the cellular error-prone capacity to repair MMS damage to DNA.
MGG Molecular & General Genetics, 1994
It has been found that the level of methyl methanesulfonate (MMS)-induced mutation in Escherichia coli is dependent on the level of UmuD(D')C proteins. The frequency of argE(ochre)--+Arg + mutations (which occur predominantly by AT--+TA transversions) and Rit s--+ Rif R mutations is much higher when UmuDC or UmuD'C are overproduced in the cell. When MMS-treated bacteria were starved for progressively longer times and hence the expression of mutations delayed, the level of mutations observed progressively declined. This same treatment had no effect on the degree of SOS induction. Examination of plasmid DNAs, isolated from MMS-treated cells, for their sensitivity to the specific endonucleases Fpg and Nth revealed that MMS causes formation of abasic sites, which are repaired during cell starvation. It is assumed that, in non-dividing cells, apurinic sites are mostly repaired by RecA-mediated recombinational repair. This pathway, which is error-free, is compared with the processing pathway in metabolically active cells, where translesion synthesis by the UmuD'2C-RecA-DNA polymerase III holoenzyme complex occurs; this latter pathway is error-prone.
Isolation, characterization, and genetic analysis of mutator genes in Escherichia coli B and K-12
Journal of Bacteriology, 1970
Twenty-one Mut mutants were obtained from Escherichia coli B (B/UV) and K-12 (JC355) after treatment with mutagens. These Mut strains are characterized by rates of mutation to streptomycin resistance and T-phase resistance which are significantly higher than the parental (Mut+) rates. Mutator genes in 12 strains have been mapped at three locations on the E. coli chromosome: one close to the leu locus; five close to the purA locus; and six close to cysC. In addition, eight mutator strains derived from E. coli B/UV are still unmapped. Some effort was made to deduce the mode of action of the mutator genes. These isolates have been examined for possible defects in deoxyribonucleic acid repair mechanisms (dark repair of ultraviolet damage, host-cell reactivation, recombination ability, repair of mitomycin C damage). By using transductional analysis, it was found that the ultraviolet sensitivity of NTG119 and its mutator property results from two separate but closely linked mutations. PurA+ transductants that receive mut from NTG119 or NTG35 are all more sensitive to mitomycin C than is the PurA recipient. Unless transduction selects for sensitivity, a probable interpretation is that defective repair of mitomycin C-induced damage is related to the mode of action of mut in these transductants and the donor. Abnormal purine synthesis may be involved in the mutability of some strains with cotransduction of the mutator property and purA (100% cotransduction for NTG119). Three mutators are recombination-deficient and may have a defective step in recombination repair. One maps near three rec genes close to cysC.
Alternative pathways of methyl methanesulfonate-induced mutagenesis in Escherichia coli
MGG Molecular & General Genetics, 1989
Methyl methanesulfonate (MMS) induced mutagenesis is known to be largely dependent on functional umuCD and reeA genes. By phenotypic analysis of Arg + (argE3, ochre) revertants according to their reversion of the mutations his-4 (ochre) and thr-1 (amber), we attempted to deduce the specificity and/or sites of MMS-induced mutations. It is shown that: (1) MMS-induced, umuC-dependent Arg + revertants (which prevail in bacteria proficient in mismatch repair) result from a different mutational pathway from umuC-independent ones. UmuC-dependent Arg + revertants belong to class 2 (Arg+His+Thr-), and umuCindependent ones to class 1 (Arg+His-Thr).
Some aspects of EMS-induced mutagenesis in Escherichia coli
Mutation Research/Reviews in Genetic Toxicology, 1993
AB2497 and its mutS and umuDC derivatives were EMS-treated at the stationary phase and specificity of mutation measured. It was found that: (i) in mutS+ cells EMS induces predominantly GC-->AT transitions (by supB or supE(oc) formation) and in mutS- cells mainly AT-->TA transversions (by supL(NG) formation); (ii) transversions of AT-->TA are umuDC-dependent and mutational specificity is biased towards AT-->GC transitions in mutS- umuDC- strains. When mutS- umuDC- cells were transfected with plasmids bearing umuD'C or umuDC genes, mutational specificity was again biased towards AT-->TA transversions; (iii) experiments with bacteria bearing umuC::lacZ or recA::lacZ fusions suggest that processing of UmuD-->UmuD' might be poorer in EMS-treated mutS- than in mutS+ cells.
Proceedings of The National Academy of Sciences, 1998
Damage-induced SOS mutagenesis requiring the UmuDC proteins occurs as part of the cells' global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD 2 C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD 2 C, activated RecA protein (RecA*), -sliding clamp, ␥-clamp loading complex,