A second DNA methyltransferase repair enzyme in Escherichia coli

Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3039-3043, May 1988 Genetics

A second DNA methyltransferase repair enzyme in Escherichia coli (ada-alB operon deletion/O'-methylguanine/04-methylthyne/suicide enzyme)

G. WILLIAM REBECK, SUSAN COONS, PATRICK CARROLL, AND LEONA SAMSON* Charles A. Dana Laboratory of Toxicology, Harvard School of Public Health, Boston, MA 02115

Communicated by Elkan R. Blout, December 28, 1987 (received for review November 9, 1987)

ada gene encodes a 39-kDa DNA methyltransferase with two active sites, one that removes methyl groups from O6methylguanine (06-MeGua) or 04-methylthymine (04MeThy) and one that removes methyl groups from methyl phosphotriester lesions (7, 12-15). The Ada protein is one of several gene products to be induced as E. coli adapt to become alkylation-resistant upon exposure to low doses of alkylating agents (3, 12, 13). The repair of methyl phosphotriester lesions converts the Ada protein into a positive regulator of the ada gene, and this adaptive response (16) and the subsequent repair of 06-MeGua and 04-MeThy lesions by the expanded pool of Ada protein prevents these lesions from surviving long enough to pass through the replication fork and generate mutations (12, 17-19). In addition, the Ada protein undergoes proteolytic cleavage to generate, from the carboxyl-terminal end of the protein, a 19-kDa methyltransferase species that can repair only 06-MeGua and 04-MeThy (7, 11, 20). The physiological role of this processing is not understood. In addition to ada, tag, and alkA, three other genes have been identified as being involved in the response of E. coli to DNA methylation damage: alkB, which forms an operon with the ada gene (21, 22); aidB, which is induced along with ada, alkB, and alkA in adapted bacteria (23); and aidC, which can be induced in response to alkylation whether or not the ada gene is functional (24). However, the function and the roles of these three gene products in the protection of E. coli against DNA alkylation damage remain unknown. Here we report that E. coli possesses another DNA methyltransferase suicide enzyme for the repair of O6MeGua and 04-MeThy, which appears to be expressed constitutively. This enzyme was identified in a deletion mutant of E. coli that lacks the entire ada-alkB operon.

The Escherichia coli ada-akB operon enABSTRACT codes a 39-kDa protein (Ada) that is a DNA-repair methyltransferase and a 27-kDa protein (AlkB) of unknown function. By DNA blot hybridization analysis we show that the alkylation-sensitive E. cofi mutant BS23 [Sedgwick, B. & Lindahl, T. (1982) J. Mol. Biol. 154, 169-1751 is a deletion mutant lacking the entire ada-aik operon. Despite the absence of the ada gene and its product, the cells contain detectable levels of a DNArepair methyltransferase activity. We conclude that the methyltransferase in BS23 cells is the product of a gene other than ada. A similar activity was detected in extracts of an ada1O::TnWO insertion mutant of E. colU AB1157. This DNA methyltransferase has a molecular mass of about 19 kDa and transfers the methyl groups from 06-methylguanine and 04methylthymine in DNA, but not those from methyl phosphotriester lesions. This enzyme was not induced by low doses of alkylating agent and is expressed at low levels in ada+ and a number of ada- E. coil strains.

The study of DNA repair and mutagenesis in Escherichia coli has uncovered intricate networks of defense mechanisms for the protection of cells against various levels of genomic damage (1). For example, two separate mechanisms operate to remove pyrimidine dimers from DNA-namely, the constitutively produced photolyase enzyme and the inducible nucleotide-excision repair pathway (2); when the level of dimers exceeds the capacity ofthese two repair pathways and threatens to cause cell death by inhibiting DNA replication, a third mechanism is induced that operates to allow E. coli to tolerate these lesions (1). In the case of DNA methylation damage, E. coli is equipped with both constitutive and inducible pathways to deal with chronic and acute exposures to methylating agents (1, 3). The inducible pathway is called the adaptive response to alkylating agents. These various constitutive and inducible enzymes mediate the repair of at least seven different types of methylated DNA lesions. The specific repair of DNA methylation damage is achieved by two types of enzymes, DNA glycosylases and DNA methyltransferases. DNA glycosylases remove certain methylated purines and pyrimidines from DNA. 3-Methyladenine DNA glycosylase I, the tag gene product, is expressed constitutively and mediates the removal of 3-methyladenine (4). 3-Methyladenine DNA glycosylase II, the product of the alkA gene, is induced as part of the adaptive response upon exposure to methylating agents (5, 6) and mediates the removal of four methylated bases-namely, 3-methyladenine, 3-methylguanine, 02-methylthymine, and 02-methylcytosine (7). If left unrepaired these four lesions are thought to present blocks to DNA replication (8), and so their removal protects E. coli from the lethal effects of DNA methylation damage (5, 6). The second type of alkylation repair enzyme, DNA methyltransferase, removes particular methyl groups from DNA in a suicide reaction that inactivates the enzyme (9-11). The

MATERIALS AND METHODS Bacterial Strains. E. coli B strains were as follows: F26 is a his- thy- derivative of E. coli B/r (25); BS21 is an adac derivative, constitutive for ada expression (26); and BS23 is an ada - derivative of BS21 (B. Sedgwick, personal communication). E. coli K-12 strains were all derivatives of AB1157: PJ3 and PJ5 are ada-3 and ada-S, respectively (27); GW5352 carries an ada-JO:: TnlO insertion (28); HK81 is nalA and HK82 is nalA alkB22 (21). BS21 and BS23 were received from P. L. Foster (Boston University), PJ3 and PJ5 were received from B. Demple (Harvard University), GW5352 was received from G. Walker (Massachusetts Institute of Technology), and HK81 and HK82 were received from Michael Volkert (University of Massachusetts, Worcester). Preparation of [3H]Methylated DNA Substrate. Micrococcus luteus DNA containing 06-[3H]MeGua as the predominant base lesion was prepared by the method of Karran et al. Abbreviations: 06-MeGua, 06-methylguanine; 0'-MeThy, 04methylthymine; MeNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MeMes, methyl methanesulfonate; MeNU, N-methyl-N-nitrosourea; adac, ada-constitutive. *To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3039

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Genetic's: Rebeck et al.

(29), using [3H]methylnitrosourea ([3H]MeNU) from Amersham (2.9 Ci/mmol; 1 Ci 37 GBq); the specific activity was 104 cpm/pzg of DNA. [3H]MeNU-methylated poly(dT)*poly(dA) substrate was prepared as described (30) and had a specific activity of 2500 cpm/fug. This substrate contained both 04-MeThy and methyl phosphotriester lesions; approximately 48% of the incorporated methyl groups were in 3-methylthymine, 42% in methyl phosphotriesters, 6% in 04-MeThy, and 4% in 02-methylthymine (30). DNA substrate containing methyl phosphotriester lesions but lacking 04-MeThy was prepared by hydrolyzing the [3H]MeNUtreated poly(dT) in 0.1 M HCl at 70'C for 30 min to remove O_-MeThy lesions (16). The hydrolysate was neutralized with 1 M NaOH and buffered to pH 8 with 0.1 volume of 1 M Tris HCI (pH 8.0). This solution was dialyzed first against 10 mM Tris-HCI, pH 7.5/1 mM EDTA/0.1 M NaCl (two changes) and then against 10 mM Tris HCl, pH 7.5/1 mM EDTA (two changes) over several days. This methylated poly(dT) was annealed to unmethylated poly(dA) to make DNA substrate lacking the 04-MeThy lesions, with a specific activity of 4200 cpm/,ug. That this substrate was lacking in 04-MeThy was confirmed by the fact that the purified 19-kDa Ada protein fragment could no longer transfer methyl groups from it (data not shown). DNA Methyltransferase Activity Gels. Cell extracts were prepared from bacteria in logarithmic growth; cells were harvested by centrifugation, the pellet was resuspended in an approximately equal volume of 50 mM Hepes-KOH, pH 7.8/10 mM dithiothreitol/l mM EDTA/5% (vol/vol) glycerol, the cells were disrupted by sonication, and the sonicate was centrifuged at 9000 x g for 15 min. The supernatants were frozen in liquid nitrogen and stored at - 70°C. One hundred micrograms of extract proteins was incubated with DNA containing particular [3H]methyl lesions for 30 min at 37°C; extracts were incubated with 19 ,ug of 06-MeGua DNA (1000 cpm), 4 ,ug of 04-MeThy/methyl phosphotriester DNA (10,000 cpm), or 2.4 ,ug of methyl phosphotriester DNA (10,000 cpm). The extract proteins were then subjected to NaDodSO4/polyacrylamide gel electrophoresis (12% acrylamide), and the gel was cut into 2-mm slices. The slices were incubated overnight at 550C in nonaqueous scintillation fluid containing 5% (vol/vol) Protosol (New England Nuclear) and then were analyzed for tritium by scintillation counting. Southern Blot Procedures. Bacterial DNA isolation (31) and Southern blot analysis (32) were carried out as described. Five micrograms of genomic DNA was digested with the indicated restriction endonucleases and the products were separated by electrophoresis in a 1% agarose gel. Blotting of the DNA onto nitrocellulose filters was by passive diffusion. The DNA fragments described in the text were labeled with 32P by nick-translation and used as probes. The final filter wash was at high stringency (30 mM NaCI/3 mM sodium citrate at 580C). Bacterial Survival Curves. Bacteria were grown at 37°C with aeration to a density of 108 cells per ml in LB medium (32). N-Methyl-N'-nitro-N-nitrosoguanidine (MeNNG; 5 ,ug/ml) or methyl methanesulfonate (MeMes; 0.05%, vol/ vol) was added, and aliquots were removed from the culture at the indicated times, diluted, and spread on LB agar plates to estimate viability. Purification of the 19-kDa Ada Fragment. Approximately 3 mg of the 19-kDa form of the Ada protein was purified to apparent homogeneity from 190 g of E. coli BS21 cells by the method of Demple et al. (11). =

RESULTS 06-MeGua DNA Methyltransferase in ada E. colt. Methyl groups transferred from alkylated DNA to the Ada methyltransferase remain associated with two cysteine residues of

Proc. Natl. Acad. Sci. USA 85 (1988) the protein (10, 11). It is therefore possible to measure DNA methyltransferase activity by incubating cell extracts with DNA containing the appropriate labeled methyl groups, followed by resolution of the proteins by NaDodSO4/polyacrylamide gel electrophoresis and identification of the labeled proteins within the gel (33). This assay allows one to determine the level and subunit molecular weight of methyltransferase activities in crude cell extracts. It has commonly been observed that ada - bacterial extracts contain a very low level of DNA methyltransferase activity, suggesting that these ada- mutants are "leaky" and express a low constitutive level of the Ada protein (34, 35). Fig. 1 shows that four different E. coli ada - strains have similar low levels of a roughly 19-kDa methyltransferase that scavenges methyl groups from DNA containing 06-MeGua; unadapted wildtype bacteria express equivalent amounts of a similar activity. The origin ofthe four ada - mutant strains was as follows: PJ3 and PJ5 were isolated from MeNNG-mutagenized E. coli AB1157 (27); GW5352 was isolated as a mini-TnlO insertion into the ada locus (28); BS23 has the Ada- phenotype and arose spontaneously from the adac strain BS21 (refs. 34 and 36; B. Sedgwick, personal communication). We were surprised to find that the ada-O:: TnlO insertion mutant, GW5352, expressed any DNA methyltransferase activity and, moreover, that the methyltransferase should appear to be of the same molecular mass as that expressed in PJ3 and PJ5, which presumably bear point mutations in the ada gene (27). These results suggested that the 19-k-Da DNA methyltransferase we observed in ada- and nonadapted E. coli might represent a second DNA methyltransferase, one that is independent of the ada gene. tIndeed, the intact 39-kDa form of the ada gene product was not detected in unadapted F26 or any ada - extracts, even though it is readily observed in extracts of the adac strain, BS21 (Fig. iF)]. Our next experiments were therefore designed to determine the level of expression of the ada gene in these mutants. If the 19-kDa DNA methyltransferase were produced in a bacterial strain 200

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FIG. 1. 06-MeGua DNA methyltransferase activity in bacterial extracts. Cell extracts (100,tg of protein) of PJ3 (A), PJ5 (B), GW5352 (C), BS23 (D), F26 (E), and BS21 (F) bacteria were incubated at 370C

for 30 mm_ with 19 jg of 0

3H]MeGua-containing DNA substrate.

After NaDodSO4/12% polyacrylamide gel electrophoresis, the location of the 3H-labeled proteins was determined by cutting the gel into 2-mm slices and eluting the proteins for liquid scintillation counting. Slice 1 is the top of the gel.

Genetics: Rebeck et al.

Proc. Natl. Acad. Sci. USA 85 (1988)

that clearly does not express the ada gene, one could infer that this enzyme is derived from a different gene. Physical Analysis of the ada-alkB Operon in ada- Strains. Gel blot analysis indicated that RNA isolated from the BS23 ada - mutant did not hybridize with either an ada or an alkB probe (data not shown). Subsequent Southern blot analysis revealed that the absence of ada-alkB mRNA was due to a deletion of the ada-alkB operon in this strain. HindIII/BamHI digests of DNA isolated from E. coli F26, BS23, BS21, GW5352, PJ3, and PJ5 were probed with a HindIII-Sma I DNA fragment that spans the entire ada gene (28). The ada probe hybridized to the expected 3.1-kilobase (kb) band (36) in every strain except BS23 (Fig. 2A). (For GW5352 DNA, the band to which the ada probe hybridizes is slightly larger than 3.1 kb, presumably as the result of the insertion of the mini-TnJO transposon.) There was no hybridization of the ada probe to BS23 DNA. When the same digests were probed with an Alu I-BamHI DNA fragment that spans the entire alkB gene (28), the alkB probe hybridized to a 3.1-kb band in every strain except, once again, BS23. Similar results were obtained when HindIII/Sma I digests of F26, BS23, BS21, and GW5352 DNA were probed with ada and alkB sequences; hybridization was observed for every strain except BS23 (data not shown). To eliminate the possibility that the ada-alkB fragments from BS23 DNA were somehow inefficiently transferred from agarose gels to nitrocellulose, we probed undigested BS23 and F26 DNA that was kb

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FIG. 2. Southern blot analysis of ada- and ada+ E. coli with ada-, alkB-, and umuC-derived sequences as probes. Five micrograms of genomic E. coli DNA was digested with HindIII and BamHI, and the resulting fragments were separated in a 1% agarose gel. Lanes 1-6: F26, BS23, BS21, GW5352, PJ3, and PJ5, respectively. Filters were hybridized to 32P-labeled ada probe (A), alkB probe (B), or umuC probe (C). Final washes were under highstringency conditions. HindIII fragments of bacteriophage A DNA were used as size markers (positions and sizes at right).

3041

directly applied to nitrocellulose for dot blot hybridization. Again, there was no hybridization of the ada or alkB probes to BS23 DNA, but there was strong hybridization to F26 DNA (data not shown). We conclude that the ada-alkB operon is deleted from E. coli BS23. As a check of the integrity of the E. coli BS23 DNA in these experiments, we probed a set of HindIII/BamHI digests with DNA from the E. coli umuDC operon, which maps 22 min away from ada (2). A mixture of two BamHI-Bgl II DNA fragments (each 1.1 kb), which were isolated from pSE117 (37) and which together span the entire umuDC operon (38), hybridized to a 9.3-kb band in every strain, including BS23 (Fig. 2C); the umuDC probe also hybridized to dot blots of both BS23 and F26 DNA (data not shown). In summary, we have found that E. coli BS23 lacks the ada-alkB operon. Since BS23, like three other ada- strains, expresses a low level of a 19-kDa DNA methyltransferase, we conclude that this enzyme is not derived from the ada gene but rather from some other gene. We propose that this enzyme be called DNA methyltransferase II. Killing of ada- Mutants by MeMes. The ada gene product provides resistance to killing by MeNNG (via the induction of the alkA gene) but does not provide substantial resistance to killing by MeMes (6, 21). The alkB gene product provides substantial resistance to killing by MeMes but not by MeNNG (21). Since both ada and alkB are deleted in E. coli BS23, these cells should be sensitive to killing by MeMes and by MeNNG, and we found that this is indeed the case (Fig. 3 A and B). Moreover, BS23 was just as sensitive to MeMes as the alkB mutant HK82 (Fig. 1C). The two strains with MeNNGinduced ada mutations, PJ3 and PJ5, which have been shown to be sensitive to MeNNG killing (27), were relatively resistant to killing by MeMes (Fig. 3B); this was shown previously for PJ5 (21). Presumably, PJ3 and PJ5 can resist killing by MeMes because the AlkB protein can be expressed adequately even though the ada gene is mutated. Interestingly, the adaJO:: TnO insertion mutant, GW5352, displayed a level of MeMes resistance intermediate between BS23 and the PJ strains, presumably because alkB is being expressed at a level higher than in BS23 but lower than in PJ3 and PJ5. Characterization of E. coli DNA Methyltransferase II. The absence of the ada gene in E. coli BS23 allowed us to determine the substrate specificity of DNA methyltransferase II. Extracts of BS23 were incubated with two alkylated DNA substrates: one carried methyl phosphotriester lesions and the other carried methyl phosphotriester plus 04-MeThy lesions (see Materials and Methods). Fig. 4 shows that methyl groups were transferred to DNA methyltransferase II only when 04-MeThy was present in the substrate. DNA methyltransferase II thus appears to be very like the 19-kDa fragment of the Ada protein, being of similar size and having the ability to accept methyl groups from 06-MeGua and 04-MeThy but not from methyl phosphotriester lesions. However, we cannot exclude the possibility that two nonAda 19-kDa methyltransferases exist, one that repairs O6MeGua and one that repairs 04-MeThy. We attempted to distinguish DNA methyltransferase II from the 19-kDa Ada protein on the basis of molecular size and reaction kinetics. The 19-kDa Ada fragment was purified to homogeneity by the method of Demple et al. (11). The enzymes had indistinguishable molecular masses as determined by NaDodSO4/polyacrylamide gel electrophoresis (data not shown). When assayed under the same reaction conditions, they transferred methyl groups from 06-MeGua at similar rates (data not shown); the purified 19-kDa fragment was assayed in the presence of crude extract prepared from BS23 cells challenged with MeNNG to deplete the endogenous DNA methyltransferase activity (see below). In addition, chromatographic analysis of amino acid hydrolysates, generated subsequent to the reaction of BS23 extracts

Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 3. MeNNG and MeMes bacterial killing curves. The colony-forming ability of various E. coli strains was measured after treatment with either MeNNG at 5 /.g/ml (A) or MeMes at 0.05% (B and C) for the indicated times. MeNNG-induced killing of F26 (ada'; l) and BS23 (.) is shown in A. MeMes-induced killing of PJ3 (n), PJ5 (i), GW5352 (o), and BS23 (*) is shown in B. MeMes-induced killing of HK82 (alkB; *) and HK81 (alkBI; n) is shown in C.

with a DNA substrate containing 06-[3H]Me Gua, indicated that, as in the case of the Ada protein, methyl groups are transferred to protein cysteine residues (dal ta not shown). Finally, we were unable to induce DNA methf yltransferase II by pretreatment of BS23 with nontoxic leve .ls of MeNNG (0.005-0.5 gg/ml). In fact, the higher pretr-eatment doses resulted in reduced methyltransferase acti'vity (data not shown). Thus, DNA methyltransferase II doe s not appear to be inducible by MeNNG. DISCUSSION 06-Alkylguanine is an extremely potent premiutagenic lesion in E. coli (12, 17-19). It is therefore not surprissing that E. coli should have evolved a number of different watys to eliminate this lesion from its genome. 06-Alkylguanine is now known to serve as substrate for at least three DNA-r epair enzymes: the uvr nucleotide-excision repair pathway has been shown to repair 06-alkylguanine lesions in vivo (ref 39; L.S., J. Thomale, and M. F. Rajewsky, unpublished data), the Ada protein removes methyl groups from 06-MelGua as well as from 0'-MeThy and methyl phosphotriester (7, 12-15), and it now seems that E. coli has a second DN)A methyltransferase that also removes methyl groups from i 06-MeGua and 04-MeThy (but not from methyl phosphottriester) but is encoded by a gene other than ada. The identiification of this second DNA methyltransferase in E. coli waas the result of our finding that the ada-alkB operon has beer deleted in the ada- strain BS23. Despite the ada deletion, I a low level of DNA methyltransferase acti 2vity. We have called this activity DNA methyltransferase I] This enzyme to appears to be constitutive and is not induced low levels of alkylating agent. The Ada protein is subject to proteolyti ic cleavage to generate a 19-kDa DNA methyltransferase species, from the

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FIG. 4. Substrate specificity of DNA methyltransf DNA BS23. Cell extracts (100 A.g of protein) were incul batedwith substrate containing methyl phosphotriester DN)A lesions (A) or methyl phosphotriester and 04-MeThy DNA lesio ons (B), and the labeled proteins were analyzed as for Fig. 1. Bovinie serum albumin (100 ,ug) was incubated with DNA substrate contaiifning both methyl phosphotriester and 04-MeThy lesions, to provi(de a measure of nonspecific transfer of radioactivity (C). re

DNA

carboxyl-terminal half of the Ada protein, that repairs O6MeGua and 0'-MeThy lesions (10, 11, 20). DNA methyltransferase II appears to be similar to the 19-kDa Ada fragment, having the same molecular size, substrate specificity, and reaction kinetics. It too transfers the methyl groups to cysteine residues. That the two methyltransferases display such similar qualities raises a question about the evolutionary relatedness of their genes. The DNA methyltransferase II gene bears little sequence homology to the ada gene, since the ada probe failed to hybridize to BS23 DNA. However, a more complete analysis of the relatedness of the two genes must await the cloning of the DNA methyltransferase II gene. It is interesting that another example of functional duplication in the repair of DNA alkylation damage is found in the two unrelated genes that code for 3-methyladenine DNA glycosylases (40). Moreover, as with the methyltransferases, one gene (tag) is expressed constitutively and the other gene (alkA) is induced as part of the adaptive response (4-6). The similarity of the 19-kDa Ada fragment and DNA methyltransferase II makes it difficult to determine their relative levels in unadapted bacteria. Mitra et al. (34) estimated that BS23 has 23 molecules of methyltransferase per cell and that wild-type F26 has 40 molecules per cell. This could suggest that about half of the DNA methyltransferase

activity in unadapted E. coli F26 can be accounted for by DNA methyltransferase II. However, in our experiments the

methyltransferase activity in BS23 and F26 were indistinguishable (about 40 molecules per cell), suggesting

levels of

cells may be that all or nearly all of the activity in that unadapted extra expression of due to DNA methyltransferase II or methyltransferase II compensates for the ada deletion. It will be interesting to determine precisely the constitutive level of Ada protein in unadapted bacteria, since the induction of the ada-alkB operon may demand a certain level of constitutive synthesis of the Ada protein. The phenotypes of the ada mutants used in the present study were quite suggestive. If the extent of MeMes resistance is related to the level of alkB expression, our results suggest that PJ3 and PJ5 express almost wild-type levels of AlkB, that GW5352 expresses somewhat lower levels of AlkB, and that BS23 expresses the lowest levels of AlkB, presumably zero. It would be surprising if alkB can be expressed at all in the ada-JO:: TnlO insertion mutant GW5352, since the alkB gene is separated from the ada-alkB 3.0 kb of of extra DNA (28). Thus, by about the expression alkB in GW5352 may itoperon seemspromoter possible that be from a promoter located within the TnlO element or from a separate alkB promoter. Indeed, Sekiguchi and coworkers (41) found evidence of a ribosome binding site and a weak promoter upstream from the alkB initiation codon at the 3' end ofthe ada gene. It would also be surprising ifPJ3 and PJ5 could

Genetics: Rebeck et al. express wild-type levels of AlkB, since in these strains the rate of ada induction is very much reduced (35). One might infer either that the timing of alkB expression is not critical for MeMes resistance or that, as already suggested, alkB can be expressed from a promoter located within the ada gene. The BS23 ada-alkB deletion mutant spontaneously arose from the adac strain BS21 that expresses high levels of the Ada protein (ref. 36; B. Sedgwick, personal communication). It appears that the continuous overexpression of Ada is unfavorable for E. coli, since ada - derivatives of BS21 arise at a rather high frequency (26); it will be interesting to determine whether all such ada - derivatives arise by deletions in this region of the chromosome. Our identification of an ada deletion in BS23 provides direct evidence that ada is not an essential gene in E. coli. However, until a mutant is identified that lacks both Ada and DNA methyltransferase II, one cannot say whether DNA methyltransferase activity is completely dispensable in E. coli. We thank C. Mark Smith for help in purifying the Ada protein fragment. We thank John Cairns, Bruce Demple, and Eric Eisenstadt for critical reading of the manuscript. This work was supported by American Cancer Society Research Grant NP448 and National Institute of Environmental Health Science Grant 1-P01-ES03926. L.S. was supported by an American Cancer Society Scholar Award and then by a Faculty Research Award. G.W.R. was supported by a National Science Foundation Graduate Research Fellowship. S.C. was supported by a National Institute of Environmental Health Sciences Graduate Training Program ES07155. P.C. was supported by a Dana Foundation Training Program for Scholars in Toxicology. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Walker, G. C. (1984) Microbiol. Rev. 48, 60-93. Friedberg, E. (1985) DNA Repair (Freeman, New York). Samson, L. & Cairns, J. (1977) Nature (London) 267, 281-283. Karran, P., Lindahl, T., Ofsteng, I., Evensen, G. B. & Seeberg, E. (1980) J. Mol. Biol. 140, 101-127. Karran, P., Hjelmgren, T. & Lindahl, T. (1982) Nature (London) 296, 770-773. Evensen, G. & Seeberg, E. (1982) Nature (London) 296, 773-775. McCarthy, T. V., Karran, P. & Lindahl, T. (1984) EMBO J. 3, 545-550. Boiteux, S., Huisman, 0. & Laval, J. (1984) EMBO J. 3, 2569-2573. Robins, P. & Cairns, J. (1979) Nature (London) 280, 74-76. Lindahl, T., Demple, B. & Robins, P. (1982) EMBO J. 1, 1359-1363. Demple, B., Jacobsson, A., Olsson, M., Robins, P. & Lindahl, T. (1982) J. Biol. Chem. 257, 13776-13780. Schendel, P. F. & Robins, P. E. (1978) Proc. NatI. Acad. Sci. USA 75, 6017-6020.

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