Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3039-3043, May 1988 Genetics A second DNA repair in (ada-alB 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 (receivedfor review November 9, 1987)

ABSTRACT The Escherichia coli ada-akB operon en- ada gene encodes a 39-kDa DNA methyltransferase with two codes a 39-kDa (Ada) that is a DNA-repair methyl- active sites, one that removes methyl groups from O6- and a 27-kDa protein (AlkB) of unknown function. methylguanine (06-MeGua) or 04-methylthymine (04- By DNA blot hybridization analysis we show that the alkyla- MeThy) and one that removes methyl groups from methyl tion-sensitive E. cofi BS23 [Sedgwick, B. & Lindahl, T. phosphotriester lesions (7, 12-15). The Ada protein is one of (1982) J. Mol. Biol. 154, 169-1751 is a deletion mutant lacking several gene products to be induced as E. coli adapt to the entire ada-aik operon. Despite the absence ofthe ada gene become alkylation-resistant upon exposure to low doses of and its product, the cells contain detectable levels of a DNA- alkylating agents (3, 12, 13). The repair of methyl phospho- repair methyltransferase activity. We conclude that the meth- triester lesions converts the Ada protein into a positive yltransferase in BS23 cells is the product of a gene other than regulator ofthe ada gene, and this adaptive response (16) and ada. A similar activity was detected in extracts of an ada- the subsequent repair of06-MeGua and 04-MeThy lesions by 1O::TnWO insertion mutant of E. colU AB1157. This DNA the expanded pool ofAda protein prevents these lesions from methyltransferase has a molecular mass of about 19 kDa and surviving long enough to pass through the replication fork transfers the methyl groups from 06-methylguanine and 04- and generate (12, 17-19). In addition, the Ada methylthymine in DNA, but not those from methyl phospho- protein undergoes proteolytic cleavage to generate, from the triester lesions. This enzyme was not induced by low doses of carboxyl-terminal end of the protein, a 19-kDa methyltrans- alkylating agent and is expressed at low levels in ada+ and a ferase species that can repair only 06-MeGua and 04-MeThy number of ada- E. coil strains. (7, 11, 20). The physiological role of this processing is not understood. The study ofDNA repair and in Escherichia coli In addition to ada, tag, and alkA, three other genes have has uncovered intricate networks of defense mechanisms for been identified as being involved in the response ofE. coli to the protection of cells against various levels of genomic DNA damage: alkB, which forms an operon with damage (1). For example, two separate mechanisms operate the ada gene (21, 22); aidB, which is induced along with ada, to remove dimers from DNA-namely, the con- alkB, and alkA in adapted (23); and aidC, which can stitutively produced photolyase enzyme and the inducible be induced in response to alkylation whether or not the ada -excision repair pathway (2); when the level of gene is functional (24). However, the function and the roles dimers exceeds the capacity ofthese two repair pathways and of these three gene products in the protection of E. coli threatens to cause cell death by inhibiting DNA replication, against DNA alkylation damage remain unknown. a third mechanism is induced that operates to allow E. coli to Here we report that E. coli possesses another DNA tolerate these lesions (1). In the case of DNA methylation methyltransferase suicide enzyme for the repair of O6- damage, E. coli is equipped with both constitutive and induci- MeGua and 04-MeThy, which appears to be expressed ble pathways to deal with chronic and acute exposures to constitutively. This enzyme was identified in a deletion methylating agents (1, 3). The inducible pathway is called the mutant of E. coli that lacks the entire ada-alkB operon. adaptive response to alkylating agents. These various consti- tutive and inducible mediate the repair of at least MATERIALS AND METHODS seven different types ofmethylated DNA lesions. The specific repair of DNA methylation damage is achieved by two types Bacterial Strains. E. coli B strains were as follows: F26 is ofenzymes, DNA glycosylases and DNA . a his- thy- derivative of E. coli B/r (25); BS21 is an adac DNA glycosylases remove certain methylated purines and derivative, constitutive for ada expression (26); and BS23 is from DNA. 3-Methyladenine DNA glycosylase an ada - derivative of BS21 (B. Sedgwick, personal commu- I, the tag gene product, is expressed constitutively and nication). E. coli K-12 strains were all derivatives ofAB1157: mediates the removal of 3-methyladenine (4). 3-Methylade- PJ3 and PJ5 are ada-3 and ada-S, respectively (27); GW5352 nine DNA glycosylase II, the product of the alkA gene, is carries an ada-JO::TnlO insertion (28); HK81 is nalA and induced as part of the adaptive response upon exposure to HK82 is nalA alkB22 (21). BS21 and BS23 were received from methylating agents (5, 6) and mediates the removal of four P. L. Foster (Boston University), PJ3 and PJ5 were received methylated bases-namely, 3-methyladenine, 3-methylgua- from B. Demple (Harvard University), GW5352 was received nine, 02-methylthymine, and 02-methylcytosine (7). If left from G. Walker (Massachusetts Institute ofTechnology), and unrepaired these four lesions are thought to present blocks to HK81 and HK82 were received from Michael Volkert (Uni- DNA replication (8), and so their removal protects E. coli versity of Massachusetts, Worcester). from the lethal effects of DNA methylation damage (5, 6). Preparation of [3H]Methylated DNA Substrate. Micrococ- The second type of alkylation repair enzyme, DNA meth- cus luteus DNA containing 06-[3H]MeGua as the predomi- yltransferase, removes particular methyl groups from DNA nant base lesion was prepared by the method of Karran et al. in a suicide reaction that inactivates the enzyme (9-11). The Abbreviations: 06-MeGua, 06-methylguanine; 0'-MeThy, 04- methylthymine; MeNNG, N-methyl-N'-nitro-N-nitrosoguanidine; The publication costs of this article were defrayed in part by page charge MeMes, methyl methanesulfonate; MeNU, N-methyl-N-nitroso- payment. This article must therefore be hereby marked "advertisement" urea; adac, ada-constitutive. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

Downloaded by guest on September 30, 2021 3039 3040 Genetic's: Rebeck et al. Proc. Natl. Acad. Sci. USA 85 (1988) (29), using [3H]methylnitrosourea ([3H]MeNU) from Amer- the protein (10, 11). It is therefore possible to measure DNA sham (2.9 Ci/mmol; 1 Ci = 37 GBq); the specific activity was methyltransferase activity by incubating cell extracts with 104 cpm/pzg of DNA. [3H]MeNU-methylated poly(dT)*poly- DNA containing the appropriate labeled methyl groups, (dA) substrate was prepared as described (30) and had a followed by resolution of the by NaDodSO4/poly- specific activity of 2500 cpm/fug. This substrate contained acrylamide gel electrophoresis and identification of the la- both 04-MeThy and methyl phosphotriester lesions; approx- beled proteins within the gel (33). This assay allows one to imately 48% of the incorporated methyl groups were in determine the level and subunit molecular weight of meth- 3-methylthymine, 42% in methyl phosphotriesters, 6% in yltransferase activities in crude cell extracts. It has com- 04-MeThy, and 4% in 02-methylthymine (30). DNA sub- monly been observed that ada - bacterial extracts contain a strate containing methyl phosphotriester lesions but lacking very low level ofDNA methyltransferase activity, suggesting 04-MeThy was prepared by hydrolyzing the [3H]MeNU- that these ada- are "leaky" and express a low treated poly(dT) in 0.1 M HCl at 70'C for 30 min to remove constitutive level of the Ada protein (34, 35). Fig. 1 shows O_-MeThy lesions (16). The hydrolysate was neutralized with that four different E. coli ada - strains have similar low levels 1 M NaOH and buffered to pH 8 with 0.1 volume of 1 M of a roughly 19-kDa methyltransferase that scavenges methyl Tris HCI (pH 8.0). This solution was dialyzed first against 10 groups from DNA containing 06-MeGua; unadapted wild- mM Tris-HCI, pH 7.5/1 mM EDTA/0.1 M NaCl (two type bacteria express equivalent amounts of a similar activ- changes) and then against 10 mM Tris HCl, pH 7.5/1 mM ity. The origin ofthe four ada- mutant strains was as follows: EDTA (two changes) over several days. This methylated PJ3 and PJ5 were poly(dT) was annealed to unmethylated poly(dA) to make isolated from MeNNG-mutagenized E. coli DNA substrate lacking the 04-MeThy lesions, with a specific AB1157 (27); GW5352 was isolated as a mini-TnlO insertion activity of 4200 cpm/,ug. That this substrate was lacking in into the ada locus (28); BS23 has the Ada- phenotype and 04-MeThy was confirmed by the fact that the purified 19-kDa arose spontaneously from the adac strain BS21 (refs. 34 and Ada protein fragment could no longer transfer methyl groups 36; B. Sedgwick, personal communication). We were sur- from it (data not shown). prised to find that the ada-O:: TnlO insertion mutant, DNA Methyltransferase Activity Gels. Cell extracts were GW5352, expressed any DNA methyltransferase activity prepared from bacteria in logarithmic growth; cells were and, moreover, that the methyltransferase should appear to harvested by centrifugation, the pellet was resuspended in an be of the same molecular mass as that expressed in PJ3 and approximately equal volume of 50 mM Hepes-KOH, pH PJ5, which presumably bear point mutations in the ada gene 7.8/10 mM dithiothreitol/l mM EDTA/5% (vol/vol) glyc- (27). These results suggested that the 19-k-Da DNA methyl- erol, the cells were disrupted by sonication, and the sonicate transferase we observed in ada- and nonadapted E. coli was centrifuged at 9000 x g for 15 min. The supernatants might represent a second DNA methyltransferase, one that is were frozen in liquid nitrogen and stored at - 70°C. One independent of the ada gene. tIndeed, the intact 39-kDa form hundred micrograms of extract proteins was incubated with of the ada gene product was not detected in unadapted F26 DNA containing particular [3H]methyl lesions for 30 min at or any ada- extracts, even though it is readily observed in 37°C; extracts were incubated with 19,ug of 06-MeGua DNA extracts of the adac strain, BS21 (Fig. iF)]. Our next (1000 cpm), 4 ,ug of04-MeThy/methyl phosphotriester DNA experiments were therefore designed to determine the level (10,000 cpm), or 2.4 ,ug of methyl phosphotriester DNA of expression of the ada gene in these mutants. If the 19-kDa (10,000 cpm). The extract proteins were then subjected to DNA methyltransferase were produced in a bacterial strain NaDodSO4/polyacrylamide gel electrophoresis (12% acryl- 200 amide), and the gel was cut into 2-mm slices. The slices were A B incubated overnight at 550C in nonaqueous scintillation fluid 150 containing 5% (vol/vol) Protosol (New England Nuclear) and then were analyzed for tritium by scintillation counting. 100 _ _ Southern Blot Procedures. Bacterial DNA isolation (31) and Southern blot analysis (32) were carried out as described. 50 I ,, Five micrograms of genomic DNA was digested with the indicated restriction and the products were C D separated by electrophoresis in a 1% agarose gel. Blotting of 1 the DNA onto nitrocellulose filters was by passive diffusion. The DNA fragments described in the text were labeled with ~)100 32P by - and used as probes. The final filter E s wash was at high stringency (30 mM NaCI/3 mM sodium 50 citrate at 580C). o Bacterial Survival Curves. Bacteria were grown at 37°C E F with aeration to a density of 108 cells per ml in LB medium 150 (32). N-Methyl-N'-nitro-N-nitrosoguanidine (MeNNG; 5 ,ug/ml) or methyl methanesulfonate (MeMes; 0.05%, vol/ 1,00 vol) was added, and aliquots were removed from the culture at the indicated times, diluted, and spread on LB agar plates 50 to estimate viability. 0 Purification of the 19-kDa Ada Fragment. Approximately 3 0 10 20 30 0 10 20 30 mg of the 19-kDa form of the Ada protein was purified to Slice number apparent homogeneity from 190 g of E. coli BS21 cells by the method of Demple et al. (11). FIG. 1. 06-MeGua DNA methyltransferase activity in bacterial extracts. Cell extracts (100,tg ofprotein) ofPJ3 (A), PJ5 (B), GW5352 (C), BS23 (D), F26 (E), and BS21 (F) bacteria were incubated at 370C RESULTS for 30 mm_ with 19 jg of 0 3H]MeGua-containing DNA substrate. After NaDodSO4/12% polyacrylamide gel electrophoresis, the loca- 06-MeGua DNA Methyltransferase in ada E. colt. Methyl tion of the3H-labeled proteins was determined by the gel into groups transferred from alkylated DNA to the Ada methyl- 2-mm slices and eluting the proteins for liquid scintillation counting. transferase remain associated with two cysteine residues of Slice 1 is the top of the gel. Downloaded by guest on September 30, 2021 Genetics: Rebeck et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3041 that clearly does not express the ada gene, one could infer directly applied to nitrocellulose for dot blot hybridization. that this enzyme is derived from a different gene. Again, there was no hybridization of the ada or alkB probes Physical Analysis of the ada-alkB Operon in ada- Strains. to BS23 DNA, but there was strong hybridization to F26 Gel blot analysis indicated that RNA isolated from the BS23 DNA (data not shown). We conclude that the ada-alkB ada - mutant did not hybridize with either an ada or an alkB operon is deleted from E. coli BS23. As a check of the probe (data not shown). Subsequent Southern blot analysis integrity of the E. coli BS23 DNA in these experiments, we revealed that the absence of ada-alkB mRNA was due to a probed a set of HindIII/BamHI digests with DNA from the deletion of the ada-alkB operon in this strain. E. coli umuDC operon, which maps 22 min away from ada HindIII/BamHI digests of DNA isolated from E. coli F26, (2). A mixture of two BamHI-Bgl II DNA fragments (each BS23, BS21, GW5352, PJ3, and PJ5 were probed with a 1.1 kb), which were isolated from pSE117 (37) and which HindIII-Sma I DNA fragment that spans the entire ada gene together span the entire umuDC operon (38), hybridized to a (28). The ada probe hybridized to the expected 3.1-kilobase 9.3-kb band in every strain, including BS23 (Fig. 2C); the (kb) band (36) in every strain except BS23 (Fig. 2A). (For umuDC probe also hybridized to dot blots of both BS23 and GW5352 DNA, the band to which the ada probe hybridizes F26 DNA (data not shown). is slightly larger than 3.1 kb, presumably as the result of the In summary, we have found that E. coli BS23 lacks the insertion of the mini-TnJO transposon.) There was no hybrid- ada-alkB operon. Since BS23, like three other ada- strains, ization of the ada probe to BS23 DNA. When the same expresses a low level of a 19-kDa DNA methyltransferase, digests were probed with an Alu I-BamHI DNA fragment we conclude that this enzyme is not derived from the ada that spans the entire alkB gene (28), the alkB probe hybrid- gene but rather from some other gene. We propose that this ized to a 3.1-kb band in every strain except, once again, enzyme be called DNA methyltransferase II. BS23. Similar results were obtained when HindIII/Sma I Killing of ada- Mutants by MeMes. The ada gene product DNA were provides resistance to killing by MeNNG (via the induction of digests of F26, BS23, BS21, and GW5352 probed the alkA gene) but does not provide substantial resistance to with ada and alkB sequences; hybridization was observed for killing by MeMes (6, 21). The alkB gene product provides every strain except BS23 (data not shown). To eliminate the substantial resistance to killing by MeMes but not by MeNNG possibility that the ada-alkB fragments from BS23 DNA were (21). Since both ada and alkB are deleted in E. coli BS23, these somehow inefficiently transferred from agarose gels to nitro- cells should be sensitive to killing by MeMes and by MeNNG, cellulose, we probed undigested BS23 and F26 DNA that was and we found that this is indeed the case (Fig. 3 A and B). kb Moreover, BS23 was just as sensitive to MeMes as the alkB A mutant HK82 (Fig. 1C). The two strains with MeNNG- -9.4 induced 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 4.3 PJ5 (21). Presumably, PJ3 and PJ5 can resist killing by MeMes because the AlkB protein can be expressed adequately even ,A464 though the ada gene is mutated. Interestingly, the ada- JO:: TnO insertion mutant, GW5352, displayed a level of -2.3 MeMes resistance intermediate between BS23 and the PJ 2 3 4 5 6 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 B absence of the ada gene in E. coli BS23 allowed us to determine the substrate specificity of DNA methyltrans- ferase II. Extracts of BS23 were incubated with two alkylated DNA substrates: one carried methyl phosphotriester lesions 4.3 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 -2.3 only when 04-MeThy was present in the substrate. DNA methyltransferase II thus appears to be very like the 19-kDa 1 2 3 4 56 fragment of the Ada protein, being of similar size and having the ability to accept methyl groups from 06-MeGua and c 04-MeThy but not from methyl phosphotriester lesions. 23.1 However, we cannot exclude the possibility that two non- Ada 19-kDa methyltransferases exist, one that repairs O6- como 94 MeGua 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 r:X - 6.5 and reaction kinetics. The 19-kDa Ada fragment was purified t, - 4.3 to homogeneity by the method of Demple et al. (11). The 2 3 4 5 6 enzymes had indistinguishable molecular masses as deter- mined by NaDodSO4/polyacrylamide gel electrophoresis FIG. 2. Southern blot analysis of ada- and ada+ E. coli with (data not shown). When assayed under the same reaction ada-, alkB-, and umuC-derived sequences as probes. Five micro- conditions, they transferred methyl groups from 06-MeGua grams of genomic E. coli DNA was digested with HindIII and at similar rates (data not shown); the purified 19-kDa frag- BamHI, and the resulting fragments were separated in a 1% agarose ment was assayed in the presence of crude extract prepared gel. Lanes 1-6: F26, BS23, BS21, GW5352, PJ3, and PJ5, respec- from cells with MeNNG to deplete the tively. Filters were hybridized to 32P-labeled ada probe (A), alkB BS23 challenged probe (B), or umuC probe (C). Final washes were under high- endogenous DNA methyltransferase activity (see below). In stringency conditions. HindIII fragments of A DNA addition, chromatographic analysis of hydroly- were used as size markers (positions and sizes at right). sates, generated subsequent to the reaction of BS23 extracts Downloaded by guest on September 30, 2021 3042 Genetics: Rebeck et al. Proc. Natl. Acad. Sci. USA 85 (1988)

~~~10-10 3 3 13 1

io-~

0 10 20 30 0 30 60 90 0 30 60 90 Time, min 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]MeGua, indicated carboxyl-terminal half of the Ada protein, that repairs O6- that, as in the case of the Ada protein, methyl groups are MeGua and 0'-MeThy lesions (10, 11, 20). DNA methyl- transferred to protein cysteine residues (dal ta not shown). transferase II appears to be similar to the 19-kDa Ada Finally, we were unable to induce DNA methf yltransferase II fragment, having the same molecular size, substrate speci- by pretreatment of BS23 with nontoxic leve .ls of MeNNG ficity, and reaction kinetics. It too transfers the methyl (0.005-0.5 gg/ml). In fact, the higher pretr-eatment doses groups to cysteine residues. That the two methyltransferases resulted in reduced methyltransferase acti'vity (data not display such similar qualities raises a question about the shown). Thus, DNA methyltransferaseII does not appear to evolutionary relatedness of their genes. The DNA methyl- be inducible by MeNNG. transferaseII gene bears little sequence homology to the ada gene, since the ada probe failed to hybridize to BS23 DNA. DISCUSSION However, a more complete analysis of the relatedness of the 06-Alkylguanine is an extremely potent premiutagenic lesion two genes must await the cloning of the DNA methyltrans- in E. coli (12, 17-19). It is therefore not surprissing that E. coli ferase II gene. It is interesting that another example of should have evolved a number of different watys to eliminate functional duplication in the repair of DNA alkylation dam- this lesion from its . 06-Alkylguanine is now known age is found in the two unrelated genes that code for to serve as substrate for at least three DNA-r epair enzymes: 3-methyladenine DNA glycosylases (40). Moreover, as with the uvr nucleotide-excision repair pathway has been shown to the methyltransferases, one gene (tag) is expressed consti- repair 06-alkylguanine lesions in vivo (ref 39; L.S., J. tutively and the other gene (alkA) is induced as part of the Thomale, and M. F. Rajewsky, unpublished data), the Ada adaptive response (4-6). protein removes methyl groups from 06-MelGua as well as The similarity of the 19-kDa Ada fragment and DNA from 0'-MeThy and methyl phosphotriester (7, 12-15), and methyltransferase II makes it difficult to determine their it now seems that E. coli has a second DN)A methyltrans- relative levels in unadapted bacteria. Mitra et al. (34) esti- ferase that also removes methyl groups fromi06-MeGua and mated that BS23 has 23 molecules of methyltransferase per 04-MeThy (but not from methyl phosphottriester) but is cell and that wild-type F26 has 40 molecules per cell. This encoded by a gene other than ada. The identiification of this could suggest that about half of the DNA methyltransferase second DNA methyltransferase in E. coli waas the result of activity in unadapted E. coli F26 can be accounted for by our finding that the ada-alkB operon has beer deleted in the DNA methyltransferase II. However, in our experiments the Despite the ada deletion, ada- strain BS23. I levels of in BS23 and F26 were a low level of DNA methyltransferase acti 2vity. We have methyltransferase activity called this activity DNA methyltransferase I] This enzyme indistinguishable (about 40 molecules per cell), suggesting that all or all of the activity in unadapted cells may be appears to be constitutive and is not induced[lThisrenzyeto due to DNAnearlymethyltransferase II or that extra expression of low levels of alkylating agent. The Ada protein is subject to proteolytiic cleavage to methyltransferase II compensates for the ada deletion. It will be interesting to determine precisely the constitutive level of generate a 19-kDa DNA methyltransferase species, from the Ada protein in unadapted bacteria, since the induction of the ada-alkB operon may demand a certain level of constitutive 200 C synthesis of the Ada protein. 150--L The phenotypes of the ada mutants used in the present I study were quite suggestive. If the extent of MeMes resis- CL L.Et tance is related to the level of alkB expression, our results suggest that PJ3 and PJ5 express almost wild-type levels of ln 50- ~Q AlkB, that GW5352 expresses somewhat lower levels of 0 1 20I - 2030 AlkB, and that BS23 expresses the lowest levels of AlkB, 0 10 20 30 0 10 20 30 0 10 20 30 presumably zero. It would be surprising if alkB can be Slice number expressed at all in the ada-JO:: TnlO insertion mutant activity in GW5352, since the alkB gene is separated from the ada-alkB FIG. 4. Substrate specificity of DNA methyltransf re DNA operon by about 3.0 kb of extra DNA (28). Thus, BS23. Cell extracts (100 A.g of protein) were incul rasedwithbatedwithDNA it seems possible that the expression of alkB in GW5352 may substrate containing methyl phosphotriester DN)A lesions (A) or or from methyl phosphotriester and 04-MeThy DNA lesioons (B), and the be from a promoter located within the TnlO element labeled proteins were analyzed as for Fig. 1. Bovinie serum albumin a separate alkB promoter. Indeed, Sekiguchi and coworkers (100 ,ug) was incubated with DNA substrate contaiifning both methyl (41) found evidence of a and a weak phosphotriester and 04-MeThy lesions, to provi(de a measure of promoter upstream from the alkB initiation codon at the 3' end nonspecific transfer of radioactivity (C). ofthe ada gene. It would also be surprising ifPJ3 and PJ5 could Downloaded by guest on September 30, 2021 Genetics: Rebeck et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3043

express wild-type levels ofAlkB, since in these strains the rate 13. Olsson, M. & Lindahl, T. (1980) J. Biol. Chem. 255, 10569- of ada induction is very much reduced (35). One might infer 10571. not 14. Foote, R. S., Mitra, S. & Pal, B. C. (1980) Biochem. Biophys. either that the timing of alkB expression is critical for Res. Commun. 97, 654-659. MeMes resistance or that, as already suggested, alkB can be 15. McCarthy, T. V. & Lindahl, T. (1985) Nucleic Acids Res. 13, expressed from a promoter located within the ada gene. 2683-2698. The BS23 ada-alkB deletion mutant spontaneously arose 16. Teo, I., Sedgwick, B., Kilpatrick, M. W., McCarthy, T. V. & from the adac strain BS21 that expresses high levels of the Lindahl, T. (1986) Cell 45, 315-324. Ada protein (ref. 36; B. Sedgwick, personal communication). 17. Loveless, A. (1%9) (London) 223, 206-207. the of Ada is 18. Dodson, L. A., Foote, R. S., Mitra, S. & Masker, W. E. (1982) It appears that continuous overexpression Proc. Natl. Acad. Sci. USA 79, 7440-7444. unfavorable for E. coli, since ada - derivatives of BS21 arise 19. Loechler, E. L., Green, C. L. & Essigman, J. M. (1984) Proc. at a rather high (26); it will be interesting to Natl. Acad. Sci. USA 81, 6271-6275. determine whether all such ada - derivatives arise by dele- 20. Teo, I., Sedgwick, B., Demple, B., Li, B. & Lindahl, T. (1984) tions in this region of the chromosome. Our identification of EMBO J. 3, 2151-2157. an ada deletion in BS23 provides direct evidence that ada is 21. Kataoka, H., Yamamoto, Y. & Sekiguchi, M. (1983) J. Bac- not an essential gene in E. coli. However, until a mutant is teriol. 153, 1301-1307. identified that lacks both Ada and DNA methyltransferase II, 22. Kataoka, H. & Sekiguchi, M. (1985) Mol. Gen. Genet. 198, 263-269. one cannot say whether DNA methyltransferase activity is 23. Volkert, M. R. & Nguyen, D. C. (1984) Proc. Natl. Acad. Sci. completely dispensable in E. coli. USA 81, 4110-4114. 24. Volkert, M. R., Nguyen, D. C. & Beard, K. C. (1986) Genetics We thank C. Mark Smith for help in purifying the Ada protein 112, 11-26. fragment. We thank John Cairns, Bruce Demple, and Eric Eisenstadt 25. Helmstetter, C. E. & Cummings, D. (1%3) Proc. Natl. Acad. for critical reading of the manuscript. This work was supported by Sci. USA 50, 767-774. American Society Research Grant NP448 and National 26. Sedgwick, B. & Robins, P. (1980) Mol. Gen. Genet. 180, 85-90. Institute of Environmental Health Science Grant 1-P01-ES03926. 27. Jeggo, P. (1979) J. Bacteriol. 139, 783-791. L.S. was supported by an American Cancer Society Scholar Award 28. LeMotte, P. K. & Walker, G. C. (1985) J. Bacteriol. 161, and then by a Faculty Research Award. G.W.R. was supported by 888-895. a National Science Foundation Graduate Research Fellowship. S.C. 29. Karran, P., Lindahl, T. & Griffin, B. (1979) Nature (London) was supported by a National Institute of Environmental Health 280, 76-77. Sciences Graduate Training Program ES07155. 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