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A Second DNA Methyltransferase Repair Enzyme in Escherichia Coli (Ada-Alb Operon Deletion/O'-Methylguanine/04-Methylthyne/Suicide Enzyme) G

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A Second DNA Methyltransferase Repair Enzyme in Escherichia Coli (Ada-Alb Operon Deletion/O'-Methylguanine/04-Methylthyne/Suicide Enzyme) G 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 (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 protein (Ada) that is a DNA-repair methyl- active sites, one that removes methyl groups from O6- transferase 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 mutant 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 mutations (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 mutagenesis 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 methylation 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 pyrimidine dimers from DNA-namely, the con- alkB, and alkA in adapted bacteria (23); and aidC, which can stitutively produced photolyase enzyme and the inducible be induced in response to alkylation whether or not the ada nucleotide-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 enzymes 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 methyltransferases. 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 pyrimidines 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 proteins 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- mutants 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).
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