Adaptive response of escherichia coli to alkylation damage

Environmental and Molecular Mutagenesis 11:241-255 (1988)

Adaptive Response of Escherichia coli to Alkylation Damage Michael R. Volkert

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester

Treatment of cells with low levels of alkylating agents for extended periods of time causes them to become resistant to the lethal and mutagenic effects of subsequent high-level challenge treatments with alkylating agents. This increased resistance has been called the adaptive response to alkylation damage and results from the induction of an alkylation-specific DNA repair response. The adaptive response is most efficiently induced by methylating agents and is most effective against the lethal and mutagenic effects of methylation damage to DNA. Four genes have been identified as components of this response, ah, alkA, alkB and aids. The functions of two of these genes are known. AlkA protein functions as a glycosylase that repairs N3-meA, N3-meG, O*-meT, and 02-meC residues in DNA, and Ada protein functions as an alkyltransferase that removes alkyl groups from 06-meG, 04-meT residues as well as methylphosphotriesters. After it interacts with methylated DNA, Ada protein functions as a positive regulatory element that controls the expression of the adaptive response by stimulating the expression of the ah-alkB operon, and the alkA and aidB genes.

Key words: DNA repair, damage-inducible genes, alkyltransferase, glycosylase, methylated DNA

INTRODUCTION

Cells at all phylogenetic levels are capable of repairing DNA damage. Their repair processes protect DNA from the consequences of damage by external agents or errors that occur during replication. The regulation and function of DNA repair mechanisms have been characterized extensively in Escherichia coli. This organism contains a variety of enzymes that repair damaged DNA. The expression of many of the genes encoding the repair enzymes is regulated and the genes are induced in response to DNA damage [for review see Kushner, 1987; Little and Mount, 1982; Singer, 1986; Walker 1984, 1985, 1987; Walker et al., 1985; Witkin, 1976, Yarosh, 19851.

The SOS genes comprise a large set of functionally diverse, damage-inducible genes that share a common regulatory mechanism [Little and Mount, 1982; Walker,

Received July 10, 1987; revised and accepted October 19, 1987.

Address reprint requests to Michael R. Volkert, Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655.

0 1988 Alan R. Liss, Inc.

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1984, 19871. The SOS response apparently constitutes a general defense against DNA damage; its general nature is suggested by the great variety of lesions that are subject to repair by this system and by the variety of agents that induce the SOS response [Little and Mount, 1982; Quillardet et al., 19821. Cells that are deficient in one or more of the SOS pathways of repair show increased sensitivity to the lethal effects of ultraviolet (UV) radiation, cross-linking agents, and many agents that produce adducts in DNA [Friedberg, 1985; Husain et al., 1985; Sancar and Rupp, 19831.

Several different types of inducible DNA repair systems have been discovered recently that are more limited with respect to the types of damaging agents that cause their induction and the types of lesions repaired. These are the adaptive response to alkylation damage and the adaptive response to oxidative damage [Demple et al., 1986; Demple and Halbrook, 1983; Samson and Cairns, 1977; Sedgwick, 1987; Sekiguchi and Nakabeppu, 19871. In this review, I will concentrate on the DNA repair mechanisms of E. coli that act on damage produced by alkylating agents, and I will discuss those features of the SOS response that are relevant to this type of damage and its repair.

GENERAL FEATURES OF THE ADAPTIVE RESPONSE TO ALKYLATION DAMAGE

The adaptive response was first characterized by Samson and Cairns [1977]. They discovered that E. coli cells grown in the presence of low levels of N-methyl- N’-nitro-N-nitrosoguanidine (MNNG) accumulated induced mutations only in the first hour. Incubation in the presence of MNNG for several days did not result in the induction of additional mutations. This suggested that the low-level exposure caused the cells to become resistant to the mutagenic effects of MNNG, that is, the cells adapted to the presence of MNNG. In further experiments cells were either adapted (or not) by incubation in the presence of MNNG for several generations. Adapted and unadapted cells were then challenged with higher doses of MNNG. Induction of the adaptive response was recognized because adapted cells were more resistant to the lethal and mutagenic effects of the challenge treatment than the unadapted cells [Samson and Cairns, 19771. A number of experiments demonstrated that the increased resistance afforded by the low-dose adaptive exposure to MNNG resulted from increased capacity to repair lesions produced by methylating agents, rather than from an alteration in permeability to the alkylating agent, or some other factor such as drug metabolism. Two results showed that the adaptive response was independent of the SOS response. It could be detected in mutants that were deficient in their ability to induce the SOS response, and some SOS inducing agents such as short wave UV radiation and 4-nitroquinoline oxide could not induce expression of the adaptive response [Jeggo et al., 1977; Schendel et al., 19781.

The requirement that cells be exposed to low levels of alkylating agents for several generations in order for induction of the adaptive response to take place is most likely due to the methods used to detect its expression. The measurement of increased resistance to lethality and mutagenesis by alkylating agents requires that cells be treated with a level of alkylating agent that is high enough to cause induction. However, an adaptive dose that is too high would kill and mutagenize enough cells to mask the reduction in lethality and mutagenesis afforded by adaptation. Therefore, only a narrow window of adaptive doses and exposure times allows detection of

Adaptive Response of E. coli 243

adaptation by such biologic measurements. Such low-dose exposures result in only a submaximal induction of the adaptive response. Greater and more rapid induction is observed when expression of lac fusions to the genes of the adaptive response is measured following exposure to doses of methylating agents considerably greater than those used in adaptation experiments [LeMotte and Walker, 1985; Nakabeppu et al., 1984b, 1985b; Otsuka et al., 1985; Volkert and Nguyen, 1984; Volkert et al., 19861.

ACTIVITIES OF METHYLATION REPAIR ENZYMES

Cells contain several enzymes whose repair activities act on alkylated bases in DNA. These enzymes function in two different pathways of DNA repair, a base excision pathway and an alkyl-group removal pathway (Fig. 1). The DNA repair enzymes that define these two pathways are quite specific with respect to the types of methylated bases they recognize.

Two N3-methyladenine (N3-meA) DNA glycosylases have been identified in E. coli. They are the products of the tug and alkA genes, respectively [Evensen and Seeberg, 1982; Nakabeppu et al., 1984a,b]. Both enzymes are capable of excising N3-me A from DNA and, like other DNA glycosylases, do so by cleaving the glycosylic bond between the N9 position of A and the C' position of the deoxyribose

Base Excision

+

3' A? sndo * 5 AP endo

P ' P T T +

Aikyl-Group Removal

b d + o b me 1.510" 06-meG in

PPPP DM

p Alkyltranrlerare

Melhylsted Alkyllranslerase

Repaired DNA

Fig. 1. A model describing the two known pathways for the repair of alkylation damage to DNA. Methylated bases and proteins are shown as filled symbols. Ada protein is shown as a five-sided polygon (see text for details). Two different lesions are shown. Base excision repairs N3-meA, N3-meG, 0'- meT, and 0'-meC. The alkyl-group removal pathway repairs 06-meG, 04-meT, and methylphosphotriesters. Only one base is shown to be replaced by polymerase for convenience. The actual size of the repair synthesis fragment is likely to be larger.

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sugar [Lindahl, 19821. The cleavage of this bond leaves an AP (apurinic/apyrimidinic) site that is a substrate for AP endonucleases. There are two types of AP endonucleases present in E. coli, one that cuts on the 3‘ side of the AP site (endonuclease In) and those that cut on the 5 ’ side of the AP site (endonuclease IV and the AP endonuclease activity of exonuclease III). Cleavage 5’ to the AP site produces a 3’ hydroxyl that primes synthesis, presumably by polymerase I [Jeggo et al., 19771. In this case the AP residue would either be removed by a 5’ AP endonuclease, or by Pol1 exonuclease activity [Warner et al., 19801. If cleavage occurs 3’ to the AP site, then the abasic sugar residue must first be removed before the 3’ hydroxyl can serve as primer for repair synthesis [Warner et al., 19801. This can be accomplished by an AP endonuclease that cuts on the 5’ side of an AP residue. After polymerization, DNA ligase can seal the nick, resulting in completion of the repair process [Lindahl, 19821.

The tug and alkA genes are regulated differently. The tug gene is constitutively expressed, and no increased activity can be detected in adapted cells. In contrast, the ulkA gene is inducible and is expressed at higher levels in adapted than in nonadapted cells [Evensen and Seeberg, 1982; Karran et al., 1982; Riazzudin and Lindahl, 19781. Moreover, the products of tag and ulkA differ in their substrate specificities. Purified preparations of the Tag enzyme excise only N3-meA; no ability to excise other methylated bases can be detected [Bjelland and Seeberg, 1987; Sakumi et al., 19861. The ulkA gene product, however, is capable of excising several different methylated bases. It reco nizes and excises N3-methylguanine (N3-meG), 02-methylcytosine (02-

[McCarthy et al., 19841. The alkyl-group removal pathway of repair is mediated by the product of the

alkyltransferase protein encoded by the ada gene (Fig. 1). This gene product was originally identified and characterized as a 19-kDa protein that repairs 06-methylguanine (06-meG) and 04-meT by transfering the methyl group from the base to itself [Demple et al., 1985; Karran et al., 1979; McCarthy et al., 1984; Olsson and Lindahl, 19801. Subsequent studies revealed that this 19-kDa protein was actually an active fragment derived by proteolysis from the 39-kDa product of the udu gene. The intact Ada protein acts as an alkyltransferase that demethylates methylphosphotriesters as well as 06-meG and 04-meT in DNA [Margison et al., 1985; McCarthy et al., 1983; McCarthy et al., 1984; McCarthy and Lindahl, 19851. Ada contains several cysteine residues, two of which act as methyl acceptors. The cysteine residue at position 321 accepts methyl groups removed from 06-meG and 04-meT (base demethylation), while the cysteine residue at position 69 serves as the acceptor for methyl groups removed from methylphosphotriesters in the sugar phosphate backbone [Demple et al., 1985; Sedgwick, 1987; Teo et al., 19861.

The alkyltransferase reaction is terminal; once a methyl acceptor site of Ada protein is occupied, it does not appear to be demethylated. Thus, Ada protein does not act enzymatically. Each molecule is able to act only once to repair a methylated base and only once to repair a methylphosphotriester.

Repair of methylphosphotriesters is stereospecific. Two stereoisomers of methylphosphotriesters, Rp and Sp, are produced in approximately equimolar amounts. The phosphotriester demethylation reaction repairs only the Sp stereoisomer; Rp forms remain unrepaired [Hamblin and Potter, 1985; McCarthy and Lindahl, 19851. The physiologic significance of this differential repair of the two stereoisomers of methylphosphotriester is unclear. This demethylation reaction may serve primarily as a regulatory rather than repair function [McCarthy and Lindahl, 19851.

meC), and 0 f -methylthymine (02-meT), in addition to N3-methyladenine (N3-meA)


Comparison of the E. coli alkyltransferase with that from other cells reveals that alkyltransferases from different sources are similar but not identical to one another. The E. coli alkyltransferase differs from the mammalian alkyltransferase from rat liver in several respects. In vitro studies show that the mammalian alkyltransferase is much less specific than the E. coli Ada protein with regard to the length of the alkyl side chain that it is capable of removing. The mammalian alkyltransferase can remove propyl groups almost as effectively as methyl groups. It can also repair butyl and isopropyl lesions, although somewhat less rapidly. However, the mammalian alkyltransferase repairs only 06-alkylguanine lesions. The repair activity of the E. coli alkyltransferase on ethyl lesions is markedly reduced compared with its activity on methyl lesions; its repair activity, however, is not limtied to the repair of lesions at the O6 position [Pegg et al., 19851.

Alkyltransferase proteins are also found in bacteria other than E. coli; however, there are differences with regard to the substrate specificity and inducibility of these repair proteins when different species are compared. Bacillus subtilis apparently contains three different alkyltransferase proteins: a constitutively expressed 06-meG alkyltransferase, an inducible 06-meG alkyltransferase, and an inducible methylphosphotriester alkyltransferase [Morohoshi and Munakata, 19871. Genetic evidence suggests that Salmonella typhimurium also contains an alkyltransferase, but experiments on adaptation in this species suggest that it is not inducible by alkylation treatment [Guttenplan and Milstein, 19821.

THE SOS RESPONSE AND ALKYLATION DAMAGE TO DNA

Cells that are deficient in the ability to induce the SOS response as a result of either a lexA or recA mutation are hypersensitive to alkylation treatment [Witkin, 1967; Yamamoto and Sekiguchi, 19791. Most alkylating agents cause induction of the SOS response [Boiteux et al., 1984; Nakabeppu et al., 1984b, 1985b; Otsuka et al., 1985; Quillardet et al., 19821. However, lesions produced by methylating agents are poor substrates for the UvrABC excision system [Warren and Lawley, 1980; Van Houton and Sancar, 19871, and mutants that lack the UvrABC excision system are no more sensitive than wild-type to methylating agents [Witkin, 19671. Only in mutants that are adu or ulkA deficient does a uvr mutation contribute to MNNG sensitivity [Van Houten and Sancar, 19871. Thus the sensitivity to methylation damage that results from recA or lexA mutations is due either to deficiencies in repair pathways other than exicision or to some indirect effect on recovery such as increased degradation of template DNA [Horan et al., 1972; Volkert et al., 19761.

As the size of the alkyl group that is added to DNA increases, the ability of the UvrABC excision repair system to excise the lesion increases [Todd and Schendel, 19831. The opposite relationship between adduct size and effectiveness of repair is seen in the case of the adaptive response. The effectiveness of MNNG adaptation in reducing the level of killing and mutagenesis that results from challenge by a second alkylating agent decreases as the size of the alkyl group added by the challenging agent increases [Jeggo et al., 1977; Todd and Schendel, 19831.

The physiologic role for the adaptive response in E. coli may be to repair those types of lesions not recognized by the UvrABC excision enzyme. Thus one view of the adaptive response to alkylating agents, and perhaps all adaptive processes, is that they are inducible DNA repair responses that render the cell better able to cope with

246 Volkert

types of DNA damage that are either poorly processed, or not processed at all, by one or more components of the SOS response.

GENETICS OF THE ADAPTIVE RESPONSE

To date three genes have been identified that contribute to the increased resistance to methylating agents that results from adaptive treatments (Fig. 2). Two of these genes, ada and aZkB, are cotranscribed from a single promoter and therefore constitute an operon [Kataoka and Sekiguchi, 1985; Kondo et al., 19861. This operon is located at 47 min on the genetic map [Jeggo, 1979; Kataoka et al., 1983; Sedgwick 19821. The third gene is aZkA, which maps at 45 min [Yamamoto et al., 1978; Volkert et al., 19861. A fourth gene, uidB (95 min), is also part of the adaptive response, but its role in survival or mutagenesis has not been established [Volkert et al., 19861.

The uda-alkB operon and the aZkA and aidB genes all share a common regulatory mechanism. Methylation-induced expression of all of these genes requires the presence of a wild-type adu gene product, indicating it functions as a regulatory element. The details of the uda-dependent regulation of the adaptive response are now reason- ably well understood and will be discussed below.

Mutations in uda, alkA, and alkB cause increased sensitivity to either the lethal or the mutagenic (or both) effects of methylation damage [Jeggo, 1979; Kataoka et al., 1983; Yamamoto et al., 19781. The sensitivities of ada and alkA mutants to alkylation damage are ascribed to their inability to repair specific lesions. No enzy- matic activity has been assigned to the aZkB gene to date, and it is not yet clear why aZkB mutants are sensitive to alkylation damage. However, since sensitivity of the alkA alkB double mutant to both MMS and MNNG is approximately the sum of the sensitivities resulting from the individual mutations, aZkA and alkB are likely to act on different lesions or function in separate pathways of repair, or both. Mutations in aidB either cause increased resistance to the lethal effects of MNNG or have no effect, depending upon the specific aidB allele [Volkert and Nguyen, 1984; Volkert et

Fig. 2. Alkylation inducible genes of E. coli. The gene symbols in boxes denote alkylation inducible genes that are regulated by Ada protein.


al., 19861. A number of possible mechanisms could account for the increased alkylation resistance resulting from some aidB mutations: decreased cell permeability, decreased metabolic activation of mutagens, or perhaps increased stability of Ada protein, which could cause increased expression of the alkA gene and the ada-alkB operon (see below).

INDUCTION OF THE ADAPTIVE RESPONSE BY DIFFERENT ALKYLATING AGENTS

When different types of alkylating agents are compared, there are two features relative to the adaptive response worthy of examination: 1) whether the lesions produced by an agent are substrates for the repair enzymes; and 2) whether the agents produce an inducing signal for the adaptive response. Adaptation of cells to MNNG offers some resistance to the lethal and mutagenic effects of ethyl and propyl derivatives of nitrosoguanidine (ENNG and PNNG respectively), indicating that the lesions produced by these alkylators are subject to repair by the enzymes induced by MNNG treatment [Todd and Schendel, 1983; Schendel et al., 19831. It is not clear from these studies what type of lesions are repaired and what enzymes carry out the repair, nor do these studies demonstrate that the resistance was the result of the induction of the ada controlled set of adaptive response genes. This question has only been addressed in the case of the ethylating agents. The udc mutants of E. coli B/r, which constitutively express the adaptive response, are more resistant than wild-type to the mutagenic effects of ENNG, EMS, and ENU, as well as MNNG, in the absence of any inducing treatment. This suggests that constitutive expression of ada controlled genes results in increased resistance to ethylation as well as methylation damage [Sedgwick and Lindahl, 19821. Consistent with this interpretation is the result that uda- mutants, which are deficient in their ability to induce the adaptive response and repair of 06- meG lesions [Demple, 19861, are more sensitive to the mutagenic effects of these ethylating agents [Sedgwick and Lindahl, 19821.

Further insights into the role of the adaptive response in repair of adducts other than methyl lesions are provided by in vitro studies of the ability of the E. coli alkyltransferase to remove different alkyl groups from the O6 position of G. This study demonstrates that purified Ada protein is very effective at removing methyl groups. It removes 50% of the methyl groups within less than 0.5 min. However, the removal rates of larger, or structurally more complex, alkyl groups are dramatically lower, requiring 10 min for the removal of 50% of the ethyl lesions and more than 90 min. for removal of 50% of the isopropyl lesions [Pegg et al., 19851. Thus the rate of dedkylation by the E. coli alkyltransferase is inversely related to the size of the alkyl group. The notion that this slow removal of ethyl and propyl groups still affords some resistance to alkylation damage by agents producing such lesions is consistent with the above observations on the effects of ada- and udc mutants on ethylation mutagenesis and killing and the fact that MNNG adaptation of wild-type increases resistance to the mutagenic effects of ethylating and propylating agents [Todd and Schendel, 1983; Schendel et al., 19831.

The second question, regarding the ability of different alkylating agents to induce the adaptive response genes, has also been addressed using fusions of the lac operon to ada or alkA. When the lac operon is transcribed from the ada or the alkA promoter in the chromosome, it is induced most efficiently by methylating agents.

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Only some ethylating agents cause induction of chromosomal aZkA-lac fusions; ENNG causes only a modest induction of alkA, while EMS and ENU cause none. The propylating agent PNNG also does not cause alkA-lac induction [Volkert, Hajec, and Gately, unpublished results]. Different results are obtained when the fusion genes are plasmid-borne. Although a plasmid-borne aZkA-ZucZ protein fusion is induced less effectively by ethylation agents than by methylating agents, ENNG, EMS, and ENU do cause induction [Nakabeppu et al., 1984b; Otsuka et al., 19851. This result suggests that expression of the alkA gene becomes relaxed when it is present on a multicopy plasmid.

One can summarize the above observations on the expression of the adaptive response as follows. The adaptive response is primarily a response to methylation damage to DNA. Although damage produced by ethylating and propylating agents is not very effective in providing an inducing signal, the lesions may serve as substrates for the repair enzymes. According to this hypothesis, cells are protected from the lethal and mutagenic effects of ethyl and propyl lesions by the basal levels of adaptive response enzymes. The additional protective effects of adaptation come about only when cells have previously been exposed to methylating agents. This feature accounts for the observation that MNNG adaptation confers resistance to challenge by ethylating and propylating agents. The possibility that additional alkylation inducible genes may exist that are not part of the adaptive response is indicated by the identification of the MNNG inducible aidC gene. The aidC gene is not dependent upon udu for its expression, and, unlike the udu controlled set of genes, aidC is effectively induced by ENNG, ENU, and PNNG [Volkert, Hajec, and Gately, unpublished results] and by other, more complex, alkylating agents [Fram, Crockett, and Volkert, unpublished results]. The role of the aidC gene in recovery from treatments with such agents is presently not understood and is complicated by the physiologic requirements for aidC expression (see below) [Fram et al., 1988; Volkert, Hajec, and Gately, unpublished results].

GENETIC REGULATION OF THE ADAPTIVE RESPONSE

Several studies have contributed to the current understanding of the regulation of the adaptive response. The identification of the gene that controls the adaptive response came from a genetic study by Jeggo [ 19791 in which colonies were screened for elevated mutation frequencies on plates containing low levels of MNNG. Mutants were found that showed reduced adaptive response induction, as measured by induced resistance to MNNG mutagenesis, killing, or both. All of these mutants mapped at one locus, called adu. Mutants constitutive for the adaptive response were found in a subsequent study by selecting for cells exhibiting increased MNU resistance [Sedg- wick and Robins, 19801. These were called udc and mapped at a position suggesting that they were also alleles of ada [Sedgwick, 19821.

The subsequent cloning of a DNA fragment containing the ada gene revealed two key features of the adaptive response. The plasmid produced a 39-kDa and a 27- kDa protein, now known to be the alkyltransferase protein and the AlkJ3 protein respectively. This study also suggested that Ada works in a positive fashion as a regulatory element [Sedgwick, 19831. This latter feature was deduced from the observation that the presence of large amounts of Ada protein in cells bearing a multicopy plasmid carrying the ada gene causes overproduction of the product of the


unlinked chromosomal aZkA gene. The positive autoregulatory activity of Ada was established by experiments with an ada-ZacZ fusion. Induction of the ada-lac2 fusion by MNNG required the presence of a second, wild-type, copy of the ada gene. When an ada-10:TnlO mutant allele was present instead of the wild-type allele, no MNNG- induced P-galactosidase activity could be detected [LeMotte and Walker, 19851. The ada-10 :TnlO allele has subsequently been shown to block induction of P-galactosidase activity of lac fusions to alkB, alkA, and aidB as well as ah-lacZ, indicating that it serves as a positive regulatory element for all of these genes [Volkert et al., 1986; Le Motte and Walker, 19851.

MOLECULAR MECHANISMS OF ada-DEPENDENT REGULATION OF GENE INDUCTION

The finding that the Ada protein was also the alkyltransferase protein suggested a possible mechanism for Ada-dependent gene regulation [Teo et al., 19841, which is outlined in Figure 3. It was proposed that methylation of the protein might activate it to cause induction of the adaptive response [Demple et al., 1985; LeMotte and Walker, 1985; Lindahl et al., 1983; Nakabeppu et al., 1985al. Biochemical studies of Ada protein have not only demonstrated this hypothesis to be correct but have led to identification of the signal that triggers the activation of Ada protein and a possible mechanism for its regulatory action.

The cloned ada and alkA genes and purified intact Ada protein were used to analyze the role of Ada in the regulation of the adaptive response in studies by Teo et al. [1986] and Nakabeppu and Sekiguchi [1986]. These two studies are in agreement that Ada protein stimulates transcription from the ada promoter only when methylated, although they disagree as to whether only methylated Ada [Teo et al., 19861 or both methylated and unmethylated Ada [Nakabeppu and Sekiguchi, 19861 are capable of stimulating transcription from the aZkA promoter. The requirement for methylation treatment for the induction of aZkA, in vivo, does not distinguish between these possibilities. It only demonstrates that, if unmethylated Ada protein can serve as an inducer of alkA, then ada protein levels must increase before induction of aZkA can occur. The general features of the model remain similar whether or not alkA responds to unmethylated Ada protein. Ada protein would be present at elevated levels only after its synthesis is induced, which requires methylation damage to DNA. However, the possible differences between the interaction of the ada and alkA promoters with Ada protein may serve to fine tune the expression of individual components of the adaptive response. The possibility that unmethylated Ada protein can serve to induce aZkA is consistent with the in vivo result that aZkA gene expression is elevated when the wild-type unmethylated Ada protein is overproduced from a high copy number plasmid carrying the ada gene [Sedgwick, 19831.

Because Ada protein can be methylated at two sites, one can hypothesize three possible mechanisms for its activation by methylation: 1) methylation of the base methyl acceptor site; 2) methylation of the phosphotriester methyl acceptor site; or 3) methylation of both sites. These possibilities were tested by Teo et al. [1986]. They found that interaction of Ada protein with synthetically constructed 06-meG containing DNA, which results in methylation of the cysteine residue at position 329 of Ada, does not activate it to stimulate transcription. Therefore, when the acceptor site for base demethylation is occupied, Ada is not activated to perform its regulatory

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Uninduced

I 1

Methylalion Damage

Ada Activation

v

Induced

1 1 1 1

+ P P Q O O O

/ I \

Fig. 3. Ada-dependent gene regulation. In all panels, the shaded symbols signify a methylated base, protein, or methylphosphotriester; the black regions signify the regulatory sequences of genes. Ada protein is shown as a five-sided polygon. Each of the two lower vertices denotes a methyl acceptor site; the left vertex is the phosphotriester methyl acceptor site, labelled P; the right, labelled B, is the base methyl acceptor site. The upper panel depicts basal level expression of the adaptive response genes. The center panel depicts the interaction of Ada protein with methylated DNA. In this panel two lesions are shown, a methylated base and a methylphosphotriester. The repair of these lesions by Ada protein results in four products: repaired DNA, the two singly methylated forms of Ada, and the doubly methylated form. The lower panel depicts the interaction of two activated forms of Ada with the operators of the &-alkB operon, and the alkA and aidB genes, resulting in induced expression of all four genes.

function. In their second experiment, synthetic poly(dA) poly(dT) was methylated, and the 04-meT lesions removed by mild acid hydrolysis. Since G residues are not present, this substrate should contain no lesions that are known to be a substrate for the base methyl acceptor site. However, this substrate does contain methylphosphotriesters. When Ada protein interacts with this type of substrate, it is activated and stimulates transcription from the ada and alkA promoters. This result indicates that Ada becomes a positive regulatory element when its phosphotriester methyl acceptor site is occupied. Moreover, this result is consistent with the in vivo observation that several mutant ada alleles that produce truncated proteins lacking the acceptor site for base demethylation, but containing a methylphosphotriester acceptor site, can still induce an ada-lacZ fusion upon MNNG treatment [LeMotte and Walker, 19851.

Both ada and alkA genes contain the DNA sequence AAANNAAAGCGCA adjacent to the presumed RNA polymerase binding site. This sequence is bound by


methylated Ada protein, as deduced from nuclease protection experiments. When methylated Ada binds to this sequence, transcription from both ada and alkA promoters is stimulated, as measured by run-off transcription assays [Nakabeppu and Sekiguchi, 1986; Teo et al., 19861. Thus the current model of regulation of the adaptive response is that Ada protein first interacts with methylated DNA, removing methyl groups from damaged bases and phosphotriesters. When its phosphotriester methyl acceptor site becomes occupied, it becomes activated and stimulates transcription of both ada and aZkA. Since wild-type Ada protein is also required for methylation-induced expression of the aidB gene [Volkert et al., 19861, Ada protein could function in the same fashion to regulate aidB.

Ada protein is readily cleaved into two fragments. The 19-kDa base alkyltransferase protein comprises the carboxyterminal half of the protein. The aminoterminal amino acid sequence of this fragment suggests that cleavage occurs between amino acids residues 178 and 179, a lysine-glutamine pair [Demple et al., 19851. The intact protein and both of the cleavage fragments are active in repair of the appropriate lesions. However, only the intact Ada protein serves as a regulatory molecule [Nakabeppu and Sekiguchi, 1986; Teo et al., 1986; B. Sedgwick, personal communi- cation]. Thus Ada cleavage is not required for its activation as a regulatory protein. Instead, once repair is completed, cleavage of Ada would result in more rapid turn- off of the adaptive response gene expression [Sedgwick, 1987; Sekiguchi and Naka- beppu, 1987; Teo et al., 19861.

THE aidC GENE

The aidC gene was identified as another alkylation inducible gene. This gene was identified as a fusion of Mu-dl(Ap'hc) that is inducible by a wide variety of alkylating agents, but not by UV. The aidC gene maps at approximately 92 min on the E. coli chromosome (Fig. 1). The sequence of genes in this region is uvrA ssb aidC, based on both genetic and physical mapping evidence [Volkert et al., 1986; Volkert and Hajec, unpublished results]. Alkylation induction of aidC is not blocked by either r e d or ada mutations, indicating that aidC is neither part of the SOS response, nor one of the ada controlled set of akylation inducible genes [Volkert et al., 1986; Volkert and Nguyen, unpublished results]. The aidC gene also differs from the ada controlled set of alkylation inducible genes in that it is strongly induced by agents that ethylate or propylate DNA [Volkert, Hajec, and Gately, unpublished results]. One curious feature of aidC induction by alkylating agents is that it is dependent upon the state of aeration. Induction of aidC is blocked by extensive aeration of the culture during alkylation treatment.

Two different links between alkylation inducible genes and the state of aeration of the culture have been observed [Fram et al., 1988; Volkert, Hajec, and Nguyen, unpublished results]. Full induction of aidC requires growth without aeration and treatment with an alkylating agent. When aidC expression is monitored by measuring P-galactosidase activity in a strain containing an aidC :Mu-dl(Ap'hc) fusion, an increase from 10 to 70 units of /I-galactosidase is seen when the culture is shifted from the aerated to the nonaerated state. More rigorous anaerobic conditions do not cause the 6-galactosidase activity to increase above this level. However, when nonaerated cells are treated with alkylating agents, P-galactosidase activities increase to a level of 300 to 400 units [Fram et al., 1988; Volkert, Hajec, and Gately, unpublished

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results]. In contrast, expression of aidB can be induced either by alkylation treatment, or by growing the cells anaerobically. Alkylation induction of aidB is &-dependent, whereas anaerobic induction is not. Anaerobic induction is unique to aidB and is not shared by other ada controlled genes [Volkert and Nguyen, 19841. It occurs when cells are grown in an atmosphere of either H2 + C 0 2 , or N2 + C02, or by growing cells to high density without aeration [Volkert, Hajec, and Nguyen, unpublished results). The relationship between anaerobiosis and alkylation treatment is currently under investigation.

FUTURE DIRECTIONS

Among the ada controlled genes, only & and alkA have known functions. The sequence of aZkB has been determined [Kondo et al., 19861; it is consistent with the production of a 27 kDa protein which is seen in maxicell extracts containing the cloned ada-aZkB operon [Kataoka and Sekiguchi, 1985; LeMotte and Walker, 1985; Sedgwick, 19831. However, further study will be required to elucidate the role of aZkB in the recovery of cells from alkylation damage.

The role of Ada proteolyses is not understood. It has not been demonstrated conclusively that this cleavage occurs in vivo, nor is it known which protease@) carries out the cleavage reaction in vitro. If this reaction proves to be physiologically relevant, then it would be interesting to learn if methylated and unmethylated forms of Ada are cleaved at different rates.

The role of aidB in alkylating agent-treated cells is also not understood, nor is it clear why this gene is also expressed at higher levels in anaerobically grown cells.

The induction of the aidC gene by a wide variety of alkylating agents demonstrates the existence of an alkylation inducible gene that is not a component of the set of genes controlled by ada. It is not known if aidC is part of a second adaptive response. How the expression of this gene is regulated and why its induction is blocked by aeration are key questions in understanding its role in cellular responses to alkylation treatment.

A link between anaerobic metabolism and recovery from alkylation damage is suggested by the dual regulation of aidB and the requirement for anaerobic conditions for alkylation induction of aidC. The nature of this link is, at present, unclear and remains an intriguing question.

ACKNOWLEDGMENTS

This work was supported by grant GM37052 from the National Institute of General Medical Sciences. I thank Zdenka Matijasevic, Laurel Hajec, Anthony Poteete, and Martin Marinus for their comments and suggestions on the manuscript.

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Adaptive response of escherichia coli to alkylation damage