Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair (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
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.
DNA Repair, 2006
The deleterious effect of defective alkB allele encoding 1meA/3meC dioxygenase on reactivation of MMS-treated phage DNA has been frequently studied. Here, it is shown that: (i) AlkB protects the cells not only against the genotoxic but also against the potent mutagenic activity of MMS; (ii) mutations arising in alkB-defected strains are umuDC-dependent, and deletion of umuDC dramatically reduce MMS-induced mutations resulting from the presence of 1meA/3meC in DNA; (iii) specificity of MMS-induced argE3-->Arg+ reversions in AB1157 alkB-defective cells are predominantly AT-->TA transversions and GC-->AT transitions; (iv) overproduction of AlkA and the resultant decrease in 3meA residues in DNA dramatically reduce MMS-induced mutations. This reduction is most probably a secondary effect of AlkA due to a decrease in 3meA residues in DNA and, in consequence, suppression of SOS induction and Pol V expression. Overproduction of UmuD'C proteins reverses this effect.
Journal of Bacteriology, 1991
Escherichia coli expresses two DNA repair methyltransferases (MTases) that repair the mutagenic 06-methylguanine (06MeG) and 04-methylthymine (04MeT) DNA lesions; one is the product of the inducible ada gene, and here we confirm that the other is the product of the constitutive ogt gene. We have generated various ogt disruption mutants. Double mutants (ada ogt) do not express any 06MeG/04MeT DNA MTases, indicating that Ada and Ogt are probably the only two 06MeG/04MeT DNA MTases in E. coli. ogt mutants were more sensitive to alkylation-induced mutation, and mutants arose linearly with dose, unlike ogt+ cells, which had a threshold dose below which no mutants accumulated; this ogt+-dependent threshold was seen in both ada' and ada strains. ogt mutants were also more sensitive to alkylation-induced killing (in an ada background), and overexpression of the Ogt MTase from a plasmid provided ada, but not ada&, cells with increased resistance to killing by alkylating agents. The induction of the adaptive response was normal in ogt mutants. We infer from these results that the Ogt MTase prevents mutagenesis by low levels of alkylating agents and that, in ada cells, the Ogt MTase also protects cells from killing by alkylating agents. We also found that ada ogt E. coli had a higher rate of spontaneous mutation than wild-type, ada, and ogt cells and that this increased mutation occurred in nondividing cells. We infer that there is an endogenous source of 06MeG or 04MeT DNA damage in E. coli that is prevalent in nondividing cells. 06-Methylguanine (O6MeG) and 04-methylthymine (O4MeT) are mutagenic DNA lesions because they can base mispair during DNA replication (18, 31). DNA methyltransferases (MTases) that repair O6MeG and O4MeT lesions irreversibly transfer methyl groups from the methylated base to specific cysteine residues in the MTase (27). MTases that repair O6MeG have been found in many organisms, including bacteria (14, 25, 40), yeasts (38), insects (11), fish (26), and mammals (3), suggesting that 06MeG DNA damage is commonly encountered. There are two known 06MeG/ 2068
Journal of bacteriology, 1989
Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did no...
Ada response - a strategy for repair of alkylated DNA in bacteria
FEMS Microbiology Letters, 2014
Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N 3 -methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N 1 -methyladenine (1meA) and N 3 -methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O 6 -methylguanine (O 6 meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA.
Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes
Proceedings of the National Academy of Sciences, 1986
The ada-alkB operon protects Escherichia coli against the effects of many alkylating agents. We have subcloned it into the pSV2 mammalian expression vector to yield pSV2ada-alkB, and this plasmid has been introduced into Mer-HeLa S3 cells, which are extremely sensitive to killing and induction of sister chromatid exchange by alkylating agents. One transformant (the S3-9 cell line) has several integrated copies of pSV2ada-alkB and was found to express a very high level of the ada gene product, the 39-kDa 06. methylguanine-DNA methyltransferase. S3-9 cells were found to have become resistant to killing and induction of sister chromatid exchange by two alkylating agents, N-methyl-N'nitro-N-nitrosoguanidine and N,N'-bis(2-chloroethyl)-N-nitrosourea. This shows that bacterial DNA alkylation-repair genes are able to suppress the alkylation-repair defects in human Mer-cells.
Journal of bacteriology, 1999
Inappropriate expression of 3-methyladenine (3MeA) DNA glycosylases has been shown to have harmful effects on microbial and mammalian cells. To understand the underlying reasons for this phenomenon, we have determined how DNA glycosylase activity and substrate specificity modulate glycosylase effects in Escherichia coli. We compared the effects of two 3MeA DNA glycosylases with very different substrate ranges, namely, the Saccharomyces cerevisiae Mag1 and the E. coli Tag glycosylases. Both glycosylases increased spontaneous mutation, decreased cell viability, and sensitized E. coli to killing by the alkylating agent methyl methanesulfonate. However, Tag had much less harmful effects than Mag1. The difference between the two enzymes' effects may be accounted for by the fact that Tag almost exclusively excises 3MeA lesions, whereas Mag1 excises a broad range of alkylated and other purines. We infer that the DNA lesions responsible for changes in spontaneous mutation, viability, an...