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.DNA alkylation damage causes mutation and cell death in both human and bacterial cells. The probability that a particular cell will die or mutate is determined by the types of DNA damage incurred and by the cellular response to the damage. Comparisons of the repair of alkylated bases in populations of alkylation-sensitive and -resistant Escherichia coli have identified 06-methylguanine as the main mutagenic lesion that causes G-C to A'T transitions (1-3) and 3-methyladenine and 3-methylguanine as lesions that lead to cell death (4-6). It is unclear whether these lesions produce the same effects in human cells. Alkylation-sensitive (Mer-) human cell lines do exist (7,8), but their phenotypes are complex and it has been difficult to use them to determine which lesions are mutagenic in human cells and which are lethal. Here we describe an approach in which we study killing and the induction of sister chromatid exchange (SCE) in Mer-human cells that have integrated the bacterial ada and alkB genes and are producing the bacterial DNA methyltransferase.The E. coli ada gene encodes a 39-kDa DNA methyltransferase that directly transfers methyl groups from o6-methylguanine, O4-methylthymine, and methyl phosphotriester DNA adducts to cysteine residues within the enzyme itself (9-12). The methyl groups remain covalently bound to the enzyme, and its DNA methyltransferase activity is consequently inactivated; i.e., the methyltransferase is a suicide enzyme (10). The alkB gene lies in the same operon as the ada gene (13). The specific function of the 27-kDa alkB gene product is not known, but it is known to be involved in the protection of bacteria from the lethal effects of alkylation damage (13, 14). We have subcloned the ada-alkB operon into a mammalian expression vector such that the operon comes under the control of the simian virus 40 (SV40) early promoter, and we have transfected this construct into alkylation-sensitive human cells.Human 10% fetal bovine serum, streptomycin (100 ,tg/ml), penicillin (100 units/ml), and L-glutamine (0.03%) and incubated in humidified 5% CO2 at 370C. G418-resistant transformants were grown in the presence of 200 ,ug ...

The effect of ATP on the first step of excision repair of ultraviolet damage in DNA has been studied using toluene-treated E. coli. During postirtadiation incubation, five to six times more single-strand breaks are formed in DNA in the presence of exogenous ATP than in its absence. The ATP-dependent as well as the ATP-independent endonucleolytic activities appear to be catalyzed by the same enzyme since both activities are almost completely absent in uvrA and uvrB mutants. An ATP-dependent endonucleolytic activity has been detected in nonirradiated toluene-treated E. coli. It is concluded that ATP is required in vivo for either the incision step of repair or an enzymatic reaction preceding it.Excision repair of UV (ultraviolet) damage in DNA is generally believed to proceed by means of an enzyme complex in which recognition of damage and chain incision, excision of damaged regions, repair replication, and rejoining of gaps occur together or in rapid succession (1, 2).Recently cells made permeable to deoxynucleoside triphosphates and other low-molecular-weight substances by treatment with toluene (3) have been used to study this repair process (4,5 Cultures were grown in M-9 medium, supplemented with 0.1% glucose, casamino acids (2.5 mg/ml), and thymidine (5 Ag/ml). Amino acids and biotin, when required, were added at 50 ,ug/ml and 1 /Ag/ml, respectively. To prelabel the bacterial chromosome, [at ]thymidine (5 gCi/ml, 5Ci/mmole) was added to the medium for three generations.Exponentially growing cells at a density of 0.5 to 1 X 109 cells per ml were harvested, resuspended at 5 X 109 cells per ml in 0.05 M potassium phosphate buffer (pH 7.4) and treated with toluene as described by Moses and Richardson (3).For each strain, duration of treatment with toluene which resulted in maximum stimulation of ATP-dependent DNA synthesis was employed. After toluenization, 2 X 109 cells per ml in 0.05 M potassium phosphate buffer (pH 7.4) were exposed to UV light (Mineralight lamp model no. R-51, maximum emission at 254 nm) at 15 ergs mm-2 sec at room temperature.Incubation mixtures (0.3 ml) contained 70 mM potassium phosphate buffer (pH 7.4), 13 mM M ++,5 mM NMN (nicotinamide mononucleotide), 2 X 108 irradiated or nonirradiated toluenized cells and ATP as indicated in the text. After incubation for 30 min at 370 in the dark, the reaction was terminated in the cold by addition of 1 ml of 6 mM EDTA (ethylenediaminetetraacetate)-0.05 M potassium phosphate buffer (pH 7.4). Lysis of cells and sedimentation analysis in alkaline sucrose gradients were performed as described byRupp and Howard-Flanders (7) with the exception that gradients were spun at 30,000 rpm for 120 min or 8,500 rpm for 18 hr at 4°. The number average molecular weight (M5) was calculated by computer from the distribution of radioactivity in each gradient, omitting only the top and bottom fractions which were clearly separated from the main radioactive peak.Phage T4 DNA (single-strand molecular weight of 55 X 106) was used as marker. It was assumed that the...

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