Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase

Hakura, Atsushi; Morimoto, Kazushige; Sofuni, Toshio; Nohmi, Takehiko

doi:10.1128/jb.173.12.3663-3672.1991

Cited by 49 publications

(32 citation statements)

References 66 publications

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“…In agreement with this, it seems that the inefficient induction of the adaptive response in S. tvphimisiiiin results from weak transcriptional activation of its ada gene by the self-methylated protein (11,39). Furthermore, chromosomal DNA sequences that hybridize with the cloned E. coli ada gene have been detected in other enterobacteria (33) which also present a poor level of induction of the alkA-lacZ fusion of E. coli.…”

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confidence: 52%

“…Adaptive repair is induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in several enterobacterial species (39), Bacillus subtilis (21,22), Bacillus thuringiensis (3), Micrococcus luteus (1), Streptomyces fradiae (2), Rhodobacter sphaeroides (40), Rhodobacter capsulatus (40), Pseudomonas aeruginosa (8), and Xanthomonas maltophilia (8). On the other hand, a similar DNA repair system has not been found in Haemophilus influenzae (16), Salmonella typhimurium (11,39,43), Neisseria gonorrhoeae (6), Rhizobium meliloti (15), or Staphylococcus au- reus (8), although a weak induction of a 39-kDa protein immunologically related to the E. coli Ada protein has been detected in S. typhimurium (39). Nevertheless, the presence of Ada-like proteins in several genera of gram-negative bacteria which were able to recognize the Ada box of E. coli has not been analyzed.…”

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confidence: 99%

“…The few enterobacteria unable to induce the alkAlacZ fusion could be such mutants, since it seems unlikely that cells taxonomically close to E. coli would have a poorly related Ada box. In this respect, the ada gene of S. typhiiniriaim has been recently cloned, and it has been shown that there is an Ada box-like sequence in its promoter region (11).…”

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Induction of the alkA gene of Escherichia coli in gram-negative bacteria

Henestrosa

Barbé

1991

J Bacteriol

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A broad-host-range plasmid containing a fusion of the alkA and lacZ genes of Escherichia coil was introduced into various aerobic and facultative gram-negative bacteria-33 species belonging to 19 genera-to study the induction of expression of the alkA gene by alkylating agents. The bacteria included species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae, and Azotobacteraceae. Results obtained show that all bacteria tested, except Aeromonas hydrophila, Agrobacterium tumefaciens, Hafnia alvei, Rhizobium meliloti, Salmonella enteritidis, Xanthomonas campestris, and those of the genus Rhodobacter, are able to induce the alkA gene of E. coli in the presence of N-methyl-N'-nitro-Nnitrosoguanidine. All these data indicate that the adaptive response to alkylating agents is present in bacterial species of several families and that the Ada box sequence must be widely conserved.

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Induction of the alkA gene of Escherichia coli in gram-negative bacteria

Henestrosa

Barbé

1991

J Bacteriol

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“…To clarify the molecular mechanism resulting in the lack of an effective adaptive response in S. typhimurium, we previously cloned an ada-like gene from S. typhimurium (ada ST ) and constructed an ada STdeletion mutant (8,60). Unlike E. coli ada mutants, which are very sensitive to the mutagenic effect of N-methyl-NЈ-nitro-Nnitrosoguanidine (MNNG) (11), the ada ST -deletion derivative exhibited almost the same sensitivity as its wild-type strain (60).…”

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Construction and characterization of mutants of Salmonella typhimurium deficient in DNA repair of O6-methylguanine

et al. 1995

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Escherichia coli has two O 6-methylguanine DNA methyltransferases that repair alkylation damage in DNA and are encoded by the ada and ogt genes. The ada gene of E. coli also regulates the adaptive response to alkylation damage. The closely related species Salmonella typhimurium possesses methyltransferase activities but does not exhibit an adaptive response conferring detectable resistance to mutagenic methylating agents. We have previously cloned the ada-like gene of S. typhimurium (ada ST ) and constructed an ada ST -deletion derivative of S. typhimurium TA1535. Unexpectedly, the sensitivity of the resulting strain to the mutagenic action of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was similar to that of the parent strain. In this study, we have cloned and sequenced the ogt-like gene of S. typhimurium (ogt ST ) and characterized ogt ST -deletion derivatives of TA1535. The ogt ST mutant was more sensitive than the parent strain to the mutagenicity of MNNG and other simple alkylating agents with longer alkyl groups (ethyl, propyl, and butyl). The ada ST -ogt ST double mutant had a level of hypersensitivity to these agents similar to that of the ogt ST single mutant. The ogt ST and the ada ST -ogt ST mutants also displayed a two to three times higher spontaneous mutation frequency than the parent strain and the ada ST mutant. These results indicate that the Ogt ST protein, but not the Ada ST protein, plays a major role in protecting S. typhimurium from the mutagenic action of endogenous as well as exogenous alkylating agents.Of the DNA lesions generated by methylating agents, a minor alkylation product O 6 -methylguanine (O 6 -MeG) is responsible for most of the mutations induced (22). This altered base directs the incorporation of either thymine or cytosine, without blocking DNA replication, resulting in GC-to-AT transition mutations (3,21,50). To counteract such mutagenic effects, most organisms possess O 6 -MeG DNA methyltransferases (MTs) that directly transfer the methyl group from O 6 -MeG to a cysteine residue within the protein in an autoinactivating stoichiometric fashion (19,20,32). The ubiquity of constitutive MTs in species ranging from bacteria to mammals (9,15,29,36,37,49,59) may indicate the occurrence of methylating agents as endogenous as well as exogenous mutagens.When Escherichia coli is exposed to subtoxic doses of methylating or ethylating agents, an adaptive response which results in the increased expression of four genes, ada, alkB, alkA, and aidB, is induced (11, 42). The ada gene encodes a 39-kDa MT which transfers the methyl group from O -ethylguanine in DNA at a rate higher than that of the Ada protein (57). The amino acid sequence of this 19-kDa protein shares homology with the C-terminal half of the 39-kDa Ada protein (4,30,35). The ogt gene is expressed constitutively (34) and is located at 29 min on the E. coli genetic map (33, 53), whereas the ada gene is located at 47 min (14,45).In contrast to the response in E. coli, an adaptive response conferring resistance to alkylating age...

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“…This indicates an approximate 2,000-fold difference at the mutagenic concentration level between the wild-type and the repairdeficient strains. Other alkylating agents also showed significant differences in mutagenic responses -methylguanine methyltransferase MGT Hakura et al, 1991;Yamada et al, 1995;, 1999 DNA TA1975 1 0 1 2 1 2 3 0 2 3 2 3 21 17 19 32 25 29 YG3002 1 2 2 8 7 8 8 21 15 92 84 88 630 1 2 3 Mean 1 2 3 Mean 1 2 3 Mean 1 2 3 Mean WP2 19 23 19 14 19 15 15 14 15 23 13 19 18 20 21 18 20 17 22 15 18 WP2uvrA 1 9 1 7 17 21 19 19 12 22 18 15 19 22 19 24 19 24 22 23 23 24 Deficient strain YG7108 YG7113 TA1535 TA1538 WP2uvrA YG3001 YG3002 YG3003 Wild strain TA1535 TA100 TA1975 TA1978 WP2 TA1535 TA1975 TA102 MNNG S9 ENNG S9 PNNG S9 BNNG S9 MMS S9 EMS S9 MNU S9 ENU …”

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Sofuni¹,

Nohmi²,

Ohta³

et al. 2005

Environ. Mutagen Res.

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SummaryThe concept of a "biological threshold" is attracting interest as an evaluation criterion for the mutagenic activity of DNA-targeting mutagens. In this context, the concept is defined as "a concentration of a chemical which does not produce any damage through its inability to perform the necessary biochemical reactions, even though present at the target in finite amount". To clarify whether this criterion is indeed applicable to DNA-targeting mutagens, we re-evaluated the reverse mutation assay data using DNA repair-deficient bacterial strains, such as S. typhimurium strains lacking the O 6 -methylguanine DNA methyltransferase genes (ada ST and ogt ST ), the nucleotide excision repair gene (uvrB) or the 8-hydroxyguanine DNA glycosylase gene (mutM ST ), and E. coli strains lacking the nucleotide excision repair gene (uvrA). Mutagenic responses of 20 test chemicals including alkylating and non-alkylating agents were compared between the repair-deficient and their wild-type strains. All the alkylating agents, such as MNNG, ENNG, EMS, ENU, DMN and DEN, exhibited more sensible mutagenic responses in strains YG7108 (∆ada ST , ∆ogt ST ) and YG7113 (same as YG7108 but containing the plasmid pKM101) than in the parental strains TA1535 and TA100 (same as TA1535 but containing the pKM101), respectively. Upon applying MNNG, YG7108 showed about 2 100 fold increase in the number of His + revertants above the spontaneous level over the range of 0.00025 0.25 µg/plate, whereas TA1535 did not show any significant increase in the number of His + revertants over the same dose range. On TA1535, an increasing tendency of the number of revertants was observed at 0.5 µg/plate or above. This indicates an approximate 2,000-fold difference at the mutagenic concentration level between the wild-type and the repairdeficient strains. Other alkylating agents also showed significant differences in mutagenic responses

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Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase

Cited by 49 publications

References 66 publications

Induction of the alkA gene of Escherichia coli in gram-negative bacteria

Induction of the alkA gene of Escherichia coli in gram-negative bacteria

Construction and characterization of mutants of Salmonella typhimurium deficient in DNA repair of O6-methylguanine

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