BIOCHIMIE, 1985, 67, 357-360 Brbve communication

The adaptive response in E. coli.

Martine DEFAIS.

Laboratoire de Pharmacologie et de Toxicologic Fondamentales, 205, route de Narbonne, 31400 Toulouse, France.

(Recue le 9-1-1985, accept~e aprEs r~vision le 20-3-1985).

R6sum6 - - La rdponse adaptative se ddveloppe dons E. coli aprOs traitement par de foibles concentrations d'agents alkylants. Ce systOme est sous le contr6le positif du gone ado. Au moins deux enzymes sont induits au cours de la rdponse : la 3-mdthyladdnine DNA glycosylase H et la 06-mdthylguanine DNA mdthyltransfdrase. Cette derniOre est aussi le produit du gone ada.

Mots-el~s : adaptation / g~ne ado / O~-m~thyignanine DNA m~thyltransf~rase / 3-m~thylad~nine DNA glycosylase II.

Summary -- The adaptive response appears in E. coli after exposure to low levels of alkylating agents. This system is under the positive control of the ada gene. At least two enzymes are induced during the response : 3-methyladenine DNA glycosylase H and 06-methylguanine DNA methyl- transferase. The latter is also the product of the ada gene.

Key-words : adaptation / ado gene / O6-methylguanine DNA methyltransferase / 3-methyladenine DNA glycosylase II.

Introduction

Alkylating agents are powerful carcinogens and mutagens. A few years ago, Samson and Cairns [1] discovered an inducible process able to repair the lesions produced in E. coli DNA by cell exposure to low concentrations of alkylating agents such as N-methyl-N'-nitro-N-nitrosogua- nidine (MNNG). The cell population then be- came resistant to the mutagenic and lethal effects of a subsequent exposure to high doses of MNNG or another alkylating agent. This repair pathway has been called the adaptive response [2], and can act on lesions produced by alkylating or ethylating agents [2, 3, 4 review 5, 6, 7]. It requires new protein synthesis [1] and has been shown to be independent of the SOS response [2].

The main mutagenic lesion produced by alky- lating agents is O6-methylguanine (O6MeG) which causes mispairing with thymine [8]. This lesion

has been shown to be specifically removed during adaptation [4, 9, 10]. 3-methyladenine is another methylation product which is removed in E. coli by DNA glycosylases, one of which is inducible during the adaptive response [15, 16, 17].

The adaptive response seems to be a relatively widespread repair system which has been identi- fied not only in several microorganisms [for review 7], but also in eukaryotes [F. Laval this volume]. In this review, I will try to summarize the status of the genetics and biochemistry of the adaptive response in Escherichia coli.

Mutants affecting adaptation

Several mutants affect the adaptive response. One series of mutations, called ado, completely blocks the response [11]. These mutants have been shown to be more sensitive to MNNG for'both killing and mutagenesis and to accumulate more

358 M. Defa~s

OrMeG than the parental strain upon long ex- posure to MNNG. The ada locus maps at 47 min on the E. coil genetic map.

Mutants constitutive for adaptation, adc mu- tants, have been isolated [3] and mapped at 47 min on the E. coli genetic map, at the same ada locus [12]. In these mutants the 06MEG lesion is hardly detectable, and they are more resistant to MNNG than their adapted parent [3]. The adc mutants contain high level of Or-methylguanine DNA methyltransferase, the enzyme responsible for the removal of mutagenic lesion. The genetic control of the methyltransferase is discussed in the last section.

Other mutants involved in the repair of lesions due to alkylating agents have been characterized. The alkA mutation which maps at 43-44 rain on the map provokes a specific sensitivity to alkyla- ting agents and a high level of mutagenesis, suggesting that the alk pathway is very accurate [14]. This mutation had previously been called tag-2 [15, 16, 17]. The alk gene product is res- ponsible for adaptation to killing by alkylating agents and needs a functional polA gene [14, 18]. The alkA gene has been shown to code for an inducible DNA glycosylase (3-methyladenine DNA glycosylase II) which releases purines methylated on the nitrogen ring: 3-methylade- nine, 3-methylguanine, 7-methyladenine and 7-methylguanine, and also O2-methylcytosine and O2-methylthymine, which were identified as im- portant lethal lesions. [16, 17, 19, 20 see last section]. The tag-1 gene codes for the constitutive 3-methyladenine DNA glycosylase activity [15, 16, 17].

Recently, the Mudl (ApR-lae) bacteriophage, which produces [3 galactosidase after insertion, was used to screen for genes induced by alkyla- ting-agents. This allowed the description of new genes of the inducible response, the a/dgenes [21]. One mutant aidA seemed to be the result of an insertion in the alkA gene. But the aidB mutants, which map in the 92-98 region, where no repair gene has been described as yet, showed an increa- sed resistance to MNNG treatment, implying that the loss of the function improves, the tolerance of the mutant to alkylating agents. An ada mutation, blocking the adaptive response, decreased [3 galactosidase synthesis in both aidA and aidB insertion mutants, suggesting a common regula- tory mechanism involving the ada gene.

Regulat ion

A great improvement in the understanding of the regulation of the adaptive response came from the cloning of the ada + gene into a cosmid vector [22]. The cosmid brought resistance to MNNG to ada mutants by allowing constitutive synthesis of high levels of both Or-methylguanine DNA methyltransferase and 3-methyladenine DNA glycosylase II, without any inducing treatment. This led to the conclusion that the ada + gene is a positive regulator of the adaptive response. Moreover the insertion of a transpos0 n in the ada locus provokes the loss of the adaptive response and the disappearance of two polypeptides of molecular weights of 37000 and 27000, which were thus identified as the products of the ada gene(s). [22, P.K. LeMotte & G.C. Walker, pers. comm.]. The two polypeptides were shown to be encoded by two adjacent genes which are coregu- lated [23, P.K. LeMotte & G.C. Walker, pers. comm.]. The 37 Kd polypeptide is the product of the regulatory gene of the adaptive response, since a plasmid carrying only this gene restores a high level of the inducible enzymes, or-methyl- guanine DNA methyltransferase and 3-methyla- denine DNA glycosylase II, in ada mutants [23]. To our knowledge, the function of the 27 Kd protein is yet to be elucidated. One possibility is that this protein represents the product of the alkB gene, which maps near the ada gene and in which mutations confer sensitivity to alkylating agents [23, 24].

An interesting observation is that antibodies raised against purified Or-methylguanine DNA methyltransferase (18 Kd) crossreact only with the 37 Kd protein [23]. In addition, Teo et al. were able to demonstrate that Or-methylguanine DNA methyltransferase is derived from the 37 Kd protein by proteolytic processing [23]. Thus the ada gene product is not only a regulatory protein, but is probably also the enzyme involved in the repair of the O6MeG mutagenic lesion. Also, a methyltransferase activity for DNA phosphotries- ters [31], induced during the adaptive response, seems to reside in the ADA protein [23].

Using an ada-lacZ operon fusion, LeMotte and Walker were able to demonstrate that truncated ADA proteins were more potent in stimulating the transcription of the ada gene than the com- plete protein; they propose that a processing of the protein is an initial step in the stimulation of its own transcription and consequently on the expression of the adaptive response. However, the nature of the inducing signal is still unknown [pers. comm.].

Adaptation in E. coil 359

Biochemis t r y o f the response

The first demonstration of a correlation bet- ween adaptation and repair of the mutagenic lesion Or-methylguanine came from the work of Schendel and Robins who demonstrated that adapted bacteria accumulated less O6-methylgua - nine in their DNA than control cells [9]. The system was saturated when too much alkylation occurred. Crude extracts from adapted cells were able to alter Ormethylguanine in DNA [25]. An enzymatic activity able to transfer methyl group from Ormethylguanine to one of its cysteine residue was purified from adapted cells and from mutants constitutive for adaptation [26, 27]. It was shown that the activity present in the adapted cells acted only once and was consumed in the reaction [27, 28]. Or-methylguanine DNA methyl- transferase has been purified to homogeneity [29]. It is a 18 Kd protein which loses its activity after the formation of the S-methylcysteine residue [30]. It is now believed that the 18 Kd protein is a still active proteolytic product of the 37 Kd protein coded by the ada gene [23].

The Or-methylguanine DNA methyltransferase has been shown recently to repair also O4-methyl - thymine [20, 31, 35]. It is interesting to note that O4-methylthymine has been reported to be a miscoding lesion [20, 36].

Among the methylation products appearing in DNA after treatment with alkylating agents, 3-methyladenine is removed rapidly [15]. The enzyme responsible is 3-methyladenine DNA glycosylase I, which is constitutively expressed. It has been purified (19 Kd protein) and released only 3-methyladenine [32]. Tag-I mutants were deficient in this enzyme but were not totally deficient in 3-methyladenine DNA glycosylase activity [16]. The residual activity represented 5-10 % of the total activity and was resistant to inhibition by free 3-methyladenine. This activity was induced during the adaptive response and existed at high levels in mutants constitutive for adaptation [16]. This 3-methyladenine DNA gly- cosylase II has been partially purified (~ 27 Kd) [33] and shown to be the product of the alkA gene [17]. It released not only 3-methyladenine and 3-methylguanine but also 7-methyladenine and 7-methylguanine. This last product was already known to be associated to the DNA glycosylase II [341.

Recently DNA glycosylase II has been shown to.catalyze the removal of two miscoding alkyla- ted pyrimidine OLmethylcytosine and O2-methyl - thymine [20, 351.

REFERENCES

I. Samson, L. & Cairns, J. (1977) Nature, 267, 281-282. 2. Jeggo, P., Defais, M., Samson, L. & Schendel, P.F.

(1977) Molec. gen. genet., 157, 1-9. 3. Sedgwick, B. & Robins, P. (1980) Molec. gen. genet.,

180, 85-90. 4. Sedgwick, B. & Lindahl, T. (1982) J. Mol. Biol., 154,

169-175. 5. Cairns, J. (1980) Nature, 286, 176-178. 6. Villani, G. (1982) Pathologie Biologie, 30, 117-123. 7. Walker, G.C. (1984) Microbiol. Rev., 48, 60-93. 8. Loveless, A. (1969) Nature, 223, 206-207. 9. Schendel, P.F. & Robins, P. (1978) Proc. Natl. Acad.

Sci. USA., 75, 6017-6020. 10. Cairns, J., Robins, P., Sedgwick, B. & Talmud, P.

(1981) Prog. Nucleic Acid Res. Mol. Biol., 26, 237-244.

11. Jeggo, P. (1979) .I. Bacteriol., 139, 783-791. 12. Sedgwick, B. (1982) J. BacterioL, 150, 984-988. 13. Yamamoto, Y., Katsuli, M., Sekiguchi, M. & Otsuji,

N. (1978) .1. Bacteriol., 135, 144-152. 14. Yamamoto, Y. & Sekiguchi, M. (1979) Molec. gen.

genet.. 171, 251-256. 15. Karran, P., Lindahl, T., Ofsteng, I., Evensen, G.B.

& Seeberg, E. (1980) J. Mol. Biol., 140, 101-127. 16. Karran, P., Hjelmgren, T. & Lindahl, T. (1982)

Nature, 296, 770-773. 17. Evensen, G. & Seeberg, E. (1982) Nature, 296,

773-775. 18. Jeggo, P.,'Defais, M., Samson, L. & Schendel, P.F.

(1978) Molec. gen. genet., 162, 299-305. 19. Yamamoto, Y., Kataoka, H., Nakabeppu, Y.,

Tsuzuki, T. & Sekiguchi, M. (1983) in : Cellular responses to DNA damage. (Friedberg E.C. & Bridges, B.A. eds) pp. 271-278, Alan R. Liss, Inc., N.Y.

20. Me Carthy, T.V., Karran, P. & LindahI, T. (1984) EMBO Journal, 3, 545-550.

21. Volkert, M.R. & Nguyen, D.C. (1984) Proc. Natl. Acad. Sci. USA, 81, 4110-4114.

22. Sedgwick, B. (1983) Molec. gen. genet., 191,466-472. 23. Teo, I., Sedgwick, B., Demple, B., Li, B. & Lindahl,

T. (1984) EMBO Journal, 3, 2151-2157. 24. Kataoka, H., Yamamoto, Y. & Sekiguchi, M. (1983)

.L BacterioL, 153, 1301-1307. 25. Karran, P., Lindahl, T. & Griffin, B. (1979) Nature,

280, 76-77. 26. Olsson, M. & Lindahl, 1". (1980) J. Biol. Chem., 255,

10569-10571. 27. Lindahl, T. (1981) in : Chromosome Damage and

Repair, (Seeberg E. & Kleppe, K., eds), pp. 207-217 Plenum Press N.Y.

28. Robins, P. & Cairns, J. (1979) Nature, 280, 74-76. 29. Demple, B., Jacobsson, A., Olsson, M., Robins, P.,

Lindahl, T. (1982) J. Biol. Chem., 257, 13776-13780.

30. Lindahl, T., Demple, B. & Robins, P. (1982) EMBO Journal, 1, 1359-1363.

360 M. Defais

31. Mc Carthy, J.G., Edington, B.V. & Schendel, P.F. (1983) Proc. NatL Acad. Sci. USA, 80, 7380-7384.

32. Riazzuddin, S. & Lindahl, T. (1978) Biochemistry, 17, 2110-2118.

33. Thomas, L., Yang, C.H. & Goldthwait, D.A. (1982) Biochemistry, 21, 1162-1169.

34. Laval, J., Pierre, J. & Laval, F. (1981) Proc. Natl. Acad. Sci USA, 78, 852.

35. Ahmed, Z. & Laval, J. (1984) Biochem. Biophys. Res. Comm., 120, 1-8.

36. Abbott, P.J. & Saffhill, R. (1977) Nucleic Acids Res., 4, 761-769.