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

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)

ABSTRACT The Escherichia coli ada-akB operon en-codes a 39-kDa protein (Ada) that is a DNA-repair methyl-transferase and a 27-kDa protein (AlkB) of unknown function.By DNA blot hybridization analysis we show that the alkyla-tion-sensitive E. cofi mutant BS23 [Sedgwick, B. & Lindahl, T.(1982) J. Mol. Biol. 154, 169-1751 is a deletion mutant lackingthe entire ada-aik operon. Despite the absence of the ada geneand its product, the cells contain detectable levels of a DNA-repair methyltransferase activity. We conclude that the meth-yltransferase in BS23 cells is the product of a gene other thanada. A similar activity was detected in extracts of an ada-1O::TnWO insertion mutant of E. colU AB1157. This DNAmethyltransferase has a molecular mass of about 19 kDa andtransfers the methyl groups from 06-methylguanine and 04-methylthymine in DNA, but not those from methyl phospho-triester lesions. This enzyme was not induced by low doses ofalkylating agent and is expressed at low levels in ada+ and anumber of ada- E. coil strains.

The study ofDNA repair and mutagenesis in Escherichia colihas uncovered intricate networks of defense mechanisms forthe protection of cells against various levels of genomicdamage (1). For example, two separate mechanisms operateto remove pyrimidine dimers from DNA-namely, the con-stitutively produced photolyase enzyme and the induciblenucleotide-excision repair pathway (2); when the level ofdimers exceeds the capacity ofthese two repair pathways andthreatens to cause cell death by inhibiting DNA replication,a third mechanism is induced that operates to allow E. coli totolerate these lesions (1). In the case of DNA methylationdamage, E. coli is equipped with both constitutive and induci-ble pathways to deal with chronic and acute exposures tomethylating agents (1, 3). The inducible pathway is called theadaptive response to alkylating agents. These various consti-tutive and inducible enzymes mediate the repair of at leastseven different types ofmethylated DNA lesions. The specificrepair ofDNA methylation damage is achieved by two typesofenzymes, DNA glycosylases and DNA methyltransferases.DNA glycosylases remove certain methylated purines and

pyrimidines from DNA. 3-Methyladenine DNA glycosylaseI, the tag gene product, is expressed constitutively andmediates the removal of 3-methyladenine (4). 3-Methylade-nine DNA glycosylase II, the product of the alkA gene, isinduced as part of the adaptive response upon exposure tomethylating agents (5, 6) and mediates the removal of fourmethylated bases-namely, 3-methyladenine, 3-methylgua-nine, 02-methylthymine, and 02-methylcytosine (7). If leftunrepaired these four lesions are thought to present blocks toDNA replication (8), and so their removal protects E. colifrom the lethal effects of DNA methylation damage (5, 6).The second type of alkylation repair enzyme, DNA meth-

yltransferase, removes particular methyl groups from DNAin a suicide reaction that inactivates the enzyme (9-11). The

ada gene encodes a 39-kDa DNA methyltransferase with twoactive sites, one that removes methyl groups from O6-methylguanine (06-MeGua) or 04-methylthymine (04-MeThy) and one that removes methyl groups from methylphosphotriester lesions (7, 12-15). The Ada protein is one ofseveral gene products to be induced as E. coli adapt tobecome alkylation-resistant upon exposure to low doses ofalkylating agents (3, 12, 13). The repair of methyl phospho-triester lesions converts the Ada protein into a positiveregulator of the ada gene, and this adaptive response (16) andthe subsequent repair of 06-MeGua and 04-MeThy lesions bythe expanded pool ofAda protein prevents these lesions fromsurviving long enough to pass through the replication forkand generate mutations (12, 17-19). In addition, the Adaprotein undergoes proteolytic cleavage to generate, from thecarboxyl-terminal end of the protein, a 19-kDa methyltrans-ferase species that can repair only 06-MeGua and 04-MeThy(7, 11, 20). The physiological role of this processing is notunderstood.

In addition to ada, tag, and alkA, three other genes havebeen identified as being involved in the response ofE. coli toDNA methylation damage: alkB, which forms an operon withthe ada gene (21, 22); aidB, which is induced along with ada,alkB, and alkA in adapted bacteria (23); and aidC, which canbe induced in response to alkylation whether or not the adagene is functional (24). However, the function and the rolesof these three gene products in the protection of E. coliagainst DNA alkylation damage remain unknown.Here we report that E. coli possesses another DNA

methyltransferase suicide enzyme for the repair of O6-MeGua and 04-MeThy, which appears to be expressedconstitutively. This enzyme was identified in a deletionmutant of E. coli that lacks the entire ada-alkB operon.

MATERIALS AND METHODSBacterial Strains. E. coli B strains were as follows: F26 is

a his- thy- derivative of E. coli B/r (25); BS21 is an adacderivative, constitutive for ada expression (26); and BS23 isan ada - derivative of BS21 (B. Sedgwick, personal commu-nication). E. coli K-12 strains were all derivatives ofAB1157:PJ3 and PJ5 are ada-3 and ada-S, respectively (27); GW5352carries an ada-JO::TnlO insertion (28); HK81 is nalA andHK82 is nalA alkB22 (21). BS21 and BS23 were received fromP. L. Foster (Boston University), PJ3 and PJ5 were receivedfrom B. Demple (Harvard University), GW5352 was receivedfrom G. Walker (Massachusetts Institute ofTechnology), andHK81 and HK82 were received from Michael Volkert (Uni-versity of Massachusetts, Worcester).

Preparation of [3H]Methylated DNA Substrate. Micrococ-cus luteus DNA containing 06-[3H]MeGua as the predomi-nant base lesion was prepared by the method of Karran et al.

Abbreviations: 06-MeGua, 06-methylguanine; 0'-MeThy, 04-methylthymine; MeNNG, N-methyl-N'-nitro-N-nitrosoguanidine;MeMes, methyl methanesulfonate; MeNU, N-methyl-N-nitroso-urea; adac, ada-constitutive.*To whom reprint requests should be addressed.

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

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(29), using [3H]methylnitrosourea ([3H]MeNU) from Amer-sham (2.9 Ci/mmol; 1 Ci = 37 GBq); the specific activity was104 cpm/pzg of DNA. [3H]MeNU-methylated poly(dT)*poly-(dA) substrate was prepared as described (30) and had aspecific activity of 2500 cpm/fug. This substrate containedboth 04-MeThy and methyl phosphotriester lesions; approx-imately 48% of the incorporated methyl groups were in3-methylthymine, 42% in methyl phosphotriesters, 6% in04-MeThy, and 4% in 02-methylthymine (30). DNA sub-strate containing methyl phosphotriester lesions but lacking04-MeThy was prepared by hydrolyzing the [3H]MeNU-treated poly(dT) in 0.1 M HCl at 70'C for 30 min to removeO_-MeThy lesions (16). The hydrolysate was neutralized with1 M NaOH and buffered to pH 8 with 0.1 volume of 1 MTris HCI (pH 8.0). This solution was dialyzed first against 10mM Tris-HCI, pH 7.5/1 mM EDTA/0.1 M NaCl (twochanges) and then against 10 mM Tris HCl, pH 7.5/1 mMEDTA (two changes) over several days. This methylatedpoly(dT) was annealed to unmethylated poly(dA) to makeDNA substrate lacking the 04-MeThy lesions, with a specificactivity of 4200 cpm/,ug. That this substrate was lacking in04-MeThy was confirmed by the fact that the purified 19-kDaAda protein fragment could no longer transfer methyl groupsfrom it (data not shown).DNA Methyltransferase Activity Gels. Cell extracts were

prepared from bacteria in logarithmic growth; cells wereharvested by centrifugation, the pellet was resuspended in anapproximately equal volume of 50 mM Hepes-KOH, pH7.8/10 mM dithiothreitol/l mM EDTA/5% (vol/vol) glyc-erol, the cells were disrupted by sonication, and the sonicatewas centrifuged at 9000 x g for 15 min. The supernatantswere frozen in liquid nitrogen and stored at - 70°C. Onehundred micrograms of extract proteins was incubated withDNA containing particular [3H]methyl lesions for 30 min at37°C; extracts were incubated with 19,ug of 06-MeGua DNA(1000 cpm), 4 ,ug of04-MeThy/methyl phosphotriester DNA(10,000 cpm), or 2.4 ,ug of methyl phosphotriester DNA(10,000 cpm). The extract proteins were then subjected toNaDodSO4/polyacrylamide gel electrophoresis (12% acryl-amide), and the gel was cut into 2-mm slices. The slices wereincubated overnight at 550C in nonaqueous scintillation fluidcontaining 5% (vol/vol) Protosol (New England Nuclear) andthen were analyzed for tritium by scintillation counting.

Southern Blot Procedures. Bacterial DNA isolation (31) andSouthern blot analysis (32) were carried out as described.Five micrograms of genomic DNA was digested with theindicated restriction endonucleases and the products wereseparated by electrophoresis in a 1% agarose gel. Blotting ofthe DNA onto nitrocellulose filters was by passive diffusion.The DNA fragments described in the text were labeled with32P by nick-translation and used as probes. The final filterwash was at high stringency (30 mM NaCI/3 mM sodiumcitrate at 580C).

Bacterial Survival Curves. Bacteria were grown at 37°Cwith 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 cultureat the indicated times, diluted, and spread on LB agar platesto estimate viability.

Purification of the 19-kDa Ada Fragment. Approximately 3mg of the 19-kDa form of the Ada protein was purified toapparent homogeneity from 190 g of E. coli BS21 cells by themethod of Demple et al. (11).

RESULTS06-MeGua DNA Methyltransferase in ada E. colt. Methyl

groups transferred from alkylated DNA to the Ada methyl-transferase remain associated with two cysteine residues of

the protein (10, 11). It is therefore possible to measure DNAmethyltransferase activity by incubating cell extracts withDNA containing the appropriate labeled methyl groups,followed by resolution of the proteins by NaDodSO4/poly-acrylamide gel electrophoresis and identification of the la-beled proteins within the gel (33). This assay allows one todetermine the level and subunit molecular weight of meth-yltransferase activities in crude cell extracts. It has com-monly been observed that ada - bacterial extracts contain avery low level ofDNA methyltransferase activity, suggestingthat these ada- mutants are "leaky" and express a lowconstitutive level of the Ada protein (34, 35). Fig. 1 showsthat four different E. coli ada - strains have similar low levelsof a roughly 19-kDa methyltransferase that scavenges methylgroups from DNA containing 06-MeGua; unadapted wild-type bacteria express equivalent amounts of a similar activ-ity. The origin ofthe four ada - mutant strains was as follows:PJ3 and PJ5 were isolated from MeNNG-mutagenized E. coliAB1157 (27); GW5352 was isolated as a mini-TnlO insertioninto the ada locus (28); BS23 has the Ada- phenotype andarose spontaneously from the adac strain BS21 (refs. 34 and36; B. Sedgwick, personal communication). We were sur-prised to find that the ada-O:: TnlO insertion mutant,GW5352, expressed any DNA methyltransferase activityand, moreover, that the methyltransferase should appear tobe of the same molecular mass as that expressed in PJ3 andPJ5, which presumably bear point mutations in the ada gene(27). These results suggested that the 19-k-Da DNA methyl-transferase we observed in ada- and nonadapted E. colimight represent a second DNA methyltransferase, one that isindependent of the ada gene. tIndeed, the intact 39-kDa formof the ada gene product was not detected in unadapted F26or any ada - extracts, even though it is readily observed inextracts of the adac strain, BS21 (Fig. iF)]. Our nextexperiments were therefore designed to determine the levelof expression of the ada gene in these mutants. If the 19-kDaDNA methyltransferase were produced in a bacterial strain

200A B

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FIG. 1. 06-MeGua DNA methyltransferase activity in bacterialextracts. Cell extracts (100,tg of protein) of PJ3 (A), PJ5 (B), GW5352(C), BS23 (D), F26 (E), and BS21 (F) bacteria were incubated at 370Cfor 30 mm_ with 19 jg of 0 3H]MeGua-containing DNA substrate.After NaDodSO4/12% polyacrylamide gel electrophoresis, the loca-tion of the 3H-labeled proteins was determined by cutting the gel into2-mm slices and eluting the proteins for liquid scintillation counting.Slice 1 is the top of the gel.

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that clearly does not express the ada gene, one could inferthat 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 BS23ada - mutant did not hybridize with either an ada or an alkBprobe (data not shown). Subsequent Southern blot analysisrevealed that the absence of ada-alkB mRNA was due to adeletion 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 aHindIII-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). (ForGW5352 DNA, the band to which the ada probe hybridizesis slightly larger than 3.1 kb, presumably as the result of theinsertion of the mini-TnJO transposon.) There was no hybrid-ization of the ada probe to BS23 DNA. When the samedigests were probed with an Alu I-BamHI DNA fragmentthat spans the entire alkB gene (28), the alkB probe hybrid-ized to a 3.1-kb band in every strain except, once again,BS23. Similar results were obtained when HindIII/Sma Idigests of F26, BS23, BS21, and GW5352 DNA were probedwith ada and alkB sequences; hybridization was observed forevery strain except BS23 (data not shown). To eliminate thepossibility that the ada-alkB fragments from BS23 DNA weresomehow inefficiently transferred from agarose gels to nitro-cellulose, we probed undigested BS23 and F26 DNA that was

kbA

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

directly applied to nitrocellulose for dot blot hybridization.Again, there was no hybridization of the ada or alkB probesto BS23 DNA, but there was strong hybridization to F26DNA (data not shown). We conclude that the ada-alkBoperon is deleted from E. coli BS23. As a check of theintegrity of the E. coli BS23 DNA in these experiments, weprobed a set of HindIII/BamHI digests with DNA from theE. coli umuDC operon, which maps 22 min away from ada(2). A mixture of two BamHI-Bgl II DNA fragments (each1.1 kb), which were isolated from pSE117 (37) and whichtogether span the entire umuDC operon (38), hybridized to a9.3-kb band in every strain, including BS23 (Fig. 2C); theumuDC probe also hybridized to dot blots of both BS23 andF26 DNA (data not shown).

In summary, we have found that E. coli BS23 lacks theada-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 adagene but rather from some other gene. We propose that thisenzyme be called DNA methyltransferase II.

Killing of ada- Mutants by MeMes. The ada gene productprovides resistance to killing by MeNNG (via the induction ofthe alkA gene) but does not provide substantial resistance tokilling by MeMes (6, 21). The alkB gene product providessubstantial resistance to killing by MeMes but not by MeNNG(21). Since both ada and alkB are deleted in E. coli BS23, thesecells 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 alkBmutant HK82 (Fig. 1C). The two strains with MeNNG-induced ada mutations, PJ3 and PJ5, which have been shownto be sensitive toMeNNG killing (27), were relatively resistantto killing by MeMes (Fig. 3B); this was shown previously forPJ5 (21). Presumably, PJ3 and PJ5 can resist killing by MeMesbecause the AlkB protein can be expressed adequately eventhough the ada gene is mutated. Interestingly, the ada-JO:: TnO insertion mutant, GW5352, displayed a level ofMeMes resistance intermediate between BS23 and the PJstrains, presumably because alkB is being expressed at a levelhigher than in BS23 but lower than in PJ3 and PJ5.

Characterization of E. coli DNA Methyltransferase II. Theabsence of the ada gene in E. coli BS23 allowed us todetermine the substrate specificity of DNA methyltrans-ferase II. Extracts of BS23 were incubated with two alkylatedDNA substrates: one carried methyl phosphotriester lesionsand the other carried methyl phosphotriester plus 04-MeThylesions (see Materials and Methods). Fig. 4 shows thatmethyl groups were transferred to DNA methyltransferase IIonly when 04-MeThy was present in the substrate. DNAmethyltransferase II thus appears to be very like the 19-kDafragment of the Ada protein, being of similar size and havingthe ability to accept methyl groups from 06-MeGua and04-MeThy but not from methyl phosphotriester lesions.However, we cannot exclude the possibility that two non-Ada 19-kDa methyltransferases exist, one that repairs O6-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 sizeand reaction kinetics. The 19-kDa Ada fragment was purifiedto homogeneity by the method of Demple et al. (11). Theenzymes had indistinguishable molecular masses as deter-mined by NaDodSO4/polyacrylamide gel electrophoresis(data not shown). When assayed under the same reactionconditions, they transferred methyl groups from 06-MeGuaat similar rates (data not shown); the purified 19-kDa frag-ment was assayed in the presence of crude extract preparedfrom BS23 cells challenged with MeNNG to deplete theendogenous DNA methyltransferase activity (see below). Inaddition, chromatographic analysis of amino acid hydroly-sates, generated subsequent to the reaction of BS23 extracts

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~~~10-10 3 3 13 1

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0 10 20 30 0 30 60 90 0 30 60 90Time, min

FIG. 3. MeNNG and MeMes bacterial killing curves. The colony-forming ability of various E. coli strains was measured after treatment witheither 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]Methat, as in the case of the Ada protein, mettransferred to protein cysteine residues (dalFinally, we were unable to induce DNA methfby pretreatment of BS23 with nontoxic leve(0.005-0.5 gg/ml). In fact, the higher pretrresulted in reduced methyltransferase acti'shown). Thus, DNA methyltransferase II doebe inducible by MeNNG.

DISCUSSION06-Alkylguanine is an extremely potent premiin E. coli (12, 17-19). It is therefore not surprisshould have evolved a number of different wathis lesion from its genome. 06-Alkylguanineto serve as substrate for at least three DNA-rthe uvr nucleotide-excision repair pathway hasrepair 06-alkylguanine lesions in vivo (refThomale, and M. F. Rajewsky, unpublishedprotein removes methyl groups from 06-Melfrom0'-MeThy and methyl phosphotriesterit now seems that E. coli has a second DN)ferase that also removes methyl groups from i04-MeThy (but not from methyl phosphotencoded by a gene other than ada. The identisecond DNA methyltransferase in E. coli waour finding that the ada-alkB operon has beerada- strain BS23. Despite the ada deletion, Ia low level of DNA methyltransferase acticalled this activity DNA methyltransferase I]appears to be constitutive and is not inducedlow levels of alkylating agent.The Ada protein is subject to proteolyti

generate a 19-kDa DNA methyltransferase sp

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FIG. 4. Substrate specificity ofDNA methyltransfBS23. Cell extracts (100 A.g of protein) were inculsubstrate containing methyl phosphotriester DN)methyl phosphotriester and 04-MeThy DNA lesiolabeled proteins were analyzed as for Fig. 1. Bovin(100 ,ug) was incubated with DNA substrate contaiiphosphotriester and 04-MeThy lesions, to provi(nonspecific transfer of radioactivity (C).

Gua, indicated carboxyl-terminal half of the Ada protein, that repairs O6-hyl groups are MeGua and 0'-MeThy lesions (10, 11, 20). DNA methyl-ta not shown). transferase II appears to be similar to the 19-kDa Adayltransferase II fragment, having the same molecular size, substrate speci-.ls of MeNNG ficity, and reaction kinetics. It too transfers the methyl-eatment doses groups to cysteine residues. That the two methyltransferasesvity (data not display such similar qualities raises a question about thes not appear to evolutionary relatedness of their genes. The DNA methyl-

transferaseII gene bears little sequence homology to the adagene, since the ada probe failed to hybridize to BS23 DNA.However, a more complete analysis of the relatedness of the

utagenic lesion two genes must await the cloning of the DNA methyltrans-sing that E. coli ferase II gene. It is interesting that another example oftys to eliminate functional duplication in the repair of DNA alkylation dam-is now known age is found in the two unrelated genes that code forepair enzymes: 3-methyladenine DNA glycosylases (40). Moreover, as withbeen shown to the methyltransferases, one gene (tag) is expressed consti-39; L.S., J. tutively and the other gene (alkA) is induced as part of the

data), the Ada adaptive response (4-6).Gua as well as The similarity of the 19-kDa Ada fragment and DNA(7, 12-15), and methyltransferase II makes it difficult to determine theirA methyltrans- relative levels in unadapted bacteria. Mitra et al. (34) esti-06-MeGua and mated that BS23 has 23 molecules of methyltransferase pertriester) but is cell and that wild-type F26 has 40 molecules per cell. Thisification of this could suggest that about half of the DNA methyltransferaseas the result of activity in unadapted E. coli F26 can be accounted for bydeleted in the DNA methyltransferase II. However, in our experiments the

2vity. We have levels of methyltransferase activity in BS23 and F26 were

This enzyme indistinguishable (about 40 molecules per cell), suggesting[lThisrenzyeto that all or nearly all of the activity in unadapted cells may be

due to DNA methyltransferase II or that extra expression of

ic cleavage to methyltransferase II compensates for the ada deletion. It willecies, from the be interesting to determine precisely the constitutive level of

Ada protein in unadapted bacteria, since the induction of theada-alkB operon may demand a certain level of constitutive

C synthesis of the Ada protein.The phenotypes of the ada mutants used in the present

study were quite suggestive. If the extent of MeMes resis-tance is related to the level of alkB expression, our resultssuggest that PJ3 and PJ5 express almost wild-type levels ofAlkB, that GW5352 expresses somewhat lower levels of

2030 AlkB, and that BS23 expresses the lowest levels of AlkB,10 20 30 presumably zero. It would be surprising if alkB can be

expressed at all in the ada-JO:: TnlO insertion mutantreactivity in GW5352, since the alkB gene is separated from the ada-alkBrasedwith DNA operon promoter by about 3.0 kb of extra DNA (28). Thus,batedwithDNA it seems possible that the expression of alkB in GW5352 mayA lesions (A) or be from a promoter located within the TnlO element or fromons (B), and theie serum albumin a separate alkB promoter. Indeed, Sekiguchi and coworkersfning both methyl (41) found evidence of a ribosome binding site and a weakde a measure of promoter upstream from the alkB initiation codon at the 3' end

ofthe ada gene. It would also be surprising ifPJ3 and PJ5 could

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express wild-type levels ofAlkB, since in these strains the rateof ada induction is very much reduced (35). One might infereither that the timing of alkB expression is not critical forMeMes resistance or that, as already suggested, alkB can beexpressed 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 theAda protein (ref. 36; B. Sedgwick, personal communication).It appears that the continuous overexpression of Ada isunfavorable for E. coli, since ada - derivatives of BS21 ariseat a rather high frequency (26); it will be interesting todetermine whether all such ada - derivatives arise by dele-tions in this region of the chromosome. Our identification ofan ada deletion in BS23 provides direct evidence that ada isnot an essential gene in E. coli. However, until a mutant isidentified that lacks both Ada and DNA methyltransferase II,one cannot say whether DNA methyltransferase activity iscompletely dispensable in E. coli.

We thank C. Mark Smith for help in purifying the Ada proteinfragment. We thank John Cairns, Bruce Demple, and Eric Eisenstadtfor critical reading of the manuscript. This work was supported byAmerican Cancer Society Research Grant NP448 and NationalInstitute of Environmental Health Science Grant 1-P01-ES03926.L.S. was supported by an American Cancer Society Scholar Awardand then by a Faculty Research Award. G.W.R. was supported bya National Science Foundation Graduate Research Fellowship. S.C.was supported by a National Institute of Environmental HealthSciences Graduate Training Program ES07155. P.C. was supportedby a Dana Foundation Training Program for Scholars in Toxicology.

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