Increased resistance to the toxic effects of alkylating agents in tobacco expressing the E. coli DNA repair gene ada.

The protein coding region of the E. coli gene ada has been transferred to tobacco plants by a leaf disc transformation procedure involving an Agrobacterium tumefaciens Ti plasmid. Transformed plants were shown to be transgenic for the ada message and had increased levels of O6-alkylguanine DNA alkyltransferase activity. The N-methyl-N-nitrosourea- or taurinechlorethylnitrosourea-induced inhibition of growth of calluses or of cells in suspension was considerably lower in ada-transformed than in non-transformed plants. This indicates that O6-alkylguanine, O4-alkylthymine or phosphotriesters are growth-inhibitory lesions in tobacco.     

Expression of the E.coli ada gene in yeast protects against the toxic and mutagenic effects of N-methyl-N''-nitro-N-nitrosoguanidine.

The E.coli ada gene protein coding region has been ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase gene promoter region to produce pADH06C. The yeast strains SX46A, 7799-4B and VV-6 are deficient in endogenous O6-alkylguanine-DNA-alkyltransferase and transformation of these strains with this shuttle vector resulted in the expression of 1730, 1260 and 374 fmoles ada-encoded ATase/mg protein in stationary phase yeast: transformation with the parent vector had no effect on endogenous ATase activity which remained less than 2 fm/mg. In comparison with parent vector transformed yeast, all of the pADH06C-transformed strains showed an increase in the resistance to the toxic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In addition, 7799-4B and VV-6 were more resistant to the mutagenic effects of this agent. These results indicate that the toxic and mutagenic effects of MNNG in yeast are mediated, at least in part, by DNA lesions than can be repaired by the E.coli ada gene product.     

Genetic mapping of ada and adc mutations affecting the adaptive response of Escherichia coli to alkylating agents.

The adaptive response to alkylating agents is an inducible repair system which protects Escherichia coli against the mutagenicity and toxicity of these agents. Four mutations, ada-3, ada-5, ada-6, and adc-1, which confer differing phenotypes as regards this response, were shown to be cotransducible with gyrA, and were located at 47 min on the E. coli genetic map. A mutation already shown on the map at 47 min as tag (B. J. Bachmann and K. B. Low, Microbiol. Rev. 44:1--56, 1980; Karran et al., J. Mol. Biol. 140:101--127, 1980) is now known to be an ada mutation (G. Evensen and E. Seeberg, personal communication).     

Exposure of E. coli to nitrosocimetidine induces the adaptive response to alkylating agents

The cytotoxic and mutagenic properties of nitrosocimetidine (NC), together with its ability to induce the adaptive response DNA-repair pathway were compared with those of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) using Escherichia coli as test organism. MNNG was found to be 250-fold more cytotoxic and 500-fold more mutagenic than NC. Prior cultivation of E. coli in low concentrations of NC protected it against the cytotoxic and/or mutagenic effects of challenge with either NC or MNNG or methyl methanesulphonate (MMS). Induction of the adaptive response by prior cultivation in low concentrations of MNNG reduced the mutagenic and cytotoxic effects of subsequent NC challenge. These results lead us to conclude that although NC is a less potent mutagen than MNNG, the DNA lesions it produces are capable of not only inducing, but also of being repaired by, the adaptive response.     

Expression of the truncated E. coli O6-methylguanine methyltransferase gene in repair-deficient human cells and restoration of cellular resistance to alkylating agents

We have constructed a truncated E. coli O6-methylguanine methyltransferase (MT) gene (ada gene) to express the MT activity for O6-methylguanine and O4-methylthymine but not for methylphosphotriester in human cells and transferred it into Mer- HeLa MR cells. The transfectant cells expressed the truncated E. coli MT were resistant to alkylating agents as same as the transfectant cells with the intact ada gene in cell killing, sister-chromatid exchange induction and host-cell reactivation of adenovirus 5. These results strongly suggest that methylphosphotriester may not contribute to the biological effect of alkylating agents in human cells.     

Cloning of the E. coli O-acetylserine sulfhydrylase gene: ability of the clone to produce a mutagenic product from azide and O-acetylserine

The gene coding for O-acetylserine sulfhydrylase (OASS) from E. coli K12 was cloned into the vector pBR322 plasmid and expressed in a cysk mutant strain of E. coli that is deficient in O-acetylserine sulfhydrylase (OASS-). The clone containing the OASS gene was selected by using tetracycline-ammonium bismuth citrate medium. Retransformation of the hybrid plasmid into competent cysk mutant cells resulted in the recovery of a clone containing normal levels of O-acetylserine sulfhydrylase. Negative selection of retransformed cysk cells on 1,2,4-triazole plates resulted in the complete inhibition of growth indicating the presence of a functional OASS gene. The ability of the new clone to convert azide to its mutagenic metabolite was tested. Cultures of the clone cells containing significant levels of OASS activity were able to produce a mutagenic product from azide and O-acetylserine as tested on Salmonella typhimurium TA1530. This cloning method could be applied also to clone the same gene from eukaryotic sources.     

Identification of protein X of Escherichia coli as the recA+/tif+ gene product.

Summary Protein X, molecular weight 40,000, has been separated from the other proteins of E. coliby a two-dimensional gel electrophoretic technique which separates proteins according to isoelectric point (pI) in the firstdimension and according to molecular weight in the second. When protein X is induced in wild-type cells by mitomycin C treatmentit has a pI6.0. However, when protein X is induced in a tif-1 mutant, either by temperatureshift-up to 42° or by mitomycin C treatment at 30°, it has a pI6.2. The low level of protein X which is present inuninduced tif mutants at 30° also has a pI6.2. These results suggest thattif-1 is a mis-sense mutation in the gene coding for protein X. Since transduction andcomplementation studies indicate that tif-1 is a mutation of therecA ⁺ gene (Castellazzi, Morand, George and Buttin, 1977) it follows that protein X is the recA ⁺ gene product.A model has been formulated to account for the relationship between protein X synthesis and the recA ⁺ and lexA ⁺ genes. In this model, a repressor coded by lexA ⁺ binds to the operator of the recA ⁺ gene from whence it can normally only be removed by the combined action of an inducer and protein X, the recA ⁺ product. Thus, protein X controls its own synthesis. The tif-1 mutation leads to a temperature sensitive form of protein X which, at 42°, can spontaneously remove the repressor without the intervention of the inducer.     

Complementation of sensitivity to alkylating agents in Escherichia coli and Chinese hamster ovary cells by expression of a cloned bacterial DNA repair gene.

Dual expression vectors derived from pSV2gpt and encoding all or part of the Escherichia coli ada+ gene have been constructed. Following transformation into an E. coli ada strain or transfection and stable integration into the genome of Chinese hamster ovary (CHO) cells, plasmid vectors containing the whole ada+ gene conferred resistance to both killing and mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Thus, the bacterial DNA repair gene was functionally expressed in the mammalian cells. Plasmids containing an N-terminal fragment of the ada+ gene which encoded only one of the two methyltransferase activities of the Ada protein did not significantly protect E. coli or CHO cells against MNNG. These results are consistent with the central role of the intact ada+ gene in controlling the adaptive response to alkylating agents in E. coli. However, the data further suggest that some alkylation lesions in DNA, such as O6-methylguanine, may exert partly different biological effects in E. coli and mammalian cells.     

Pathways of mutagenesis and repair in Escherichia coli exposed to low levels of simple alkylating agents.

Mutagenesis by simple alkylating agents is thought to occur by either a lexA+-dependent process called error-prone repair or a lex-independent process often attributed to mispairing during replication. We show here that error-prone repair is responsible for the majority of mutants formed after a large dose of alkylating agent, but it is unlikely that it contributes significantly to mutagenesis during exposure to low concentrations of these chemicals. The mutagenicity of these low doses of alkylating agent is reduced by a repair system constitutively present in lexA+ cells but absent in lexA mutants. This system reduces mutagenesis until a second error-free system, called the adaptive responses, can be induced [P. Jeggo, M. Defais, L. Samson, and P. Schendel, Mol. Gen. Genet, 157:1-9, 1977; L. Samson and J. Cairns, Nature (London) 267:281-283, 1977]. The adaptive response is capable of dealing with a much larger amount of alkylation damage than the constitutive system and, when induced, appears to be able to reduce mutagenesis by both decreasing the number of sites available for mutagenesis and delaying the induction of error-prone repair enzymes. Finally, we discuss a model of chemically induced mutagenesis based on these findings which maintains that the observed mutation frequency is dependent on a "race" between these two error-free systems and the two mutagenic pathways.     

Identification of the Escherichia coli recN gene product as a major SOS protein.

The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified.