Exposure of Escherichia coli to simple alkylating agents, such as methylnitrosourea, causes the induction of at least three DNA repair functions that are under the control of the ada gene. The ada gene product itself repairs several O-methylated lesions in DNA, including methylphosphotriesters and the mutagenic adduct O1-methylguanine. The methyl groups are transferred from these lesions on to two different cysteine residues within the Ada protein resulting in selfmethylation. We have found that the Ada protein is converted to an activator of expression of genes involved in the adaptive response after accepting a methyl group from a methylphosphotriester, but not from O1-methylguanine. This was shown using the in vitro techniques of DNA-dependent protein synthesis and run-off transcription. Delayed electrophoretic migration and footprinting experiments have shown that the methylated activator of transcription binds to specific DNA sequences immediately upstream from the RNA polymerase binding sites in the promoter regions of the inducible genes. The Ada protein-binding sites contain the common sequence d(A-A-A-N- N-A-A-A-G-C-G-C-A).

The adaptive response to alkylation damage is induced in Escherichia coli on exposure to simple alkylating agents, such as jV-methylnitrosourea (MNU) and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) (Samson & Cairns, 1977). This DNA repair pathway is not induced by ultraviolet irradiation and occurs in RecA mutants, and is therefore independent of the inducible SOS response (Jeggo et al. 1977). The increased resistance of the induced cells to both the mutagenic and toxic effects of alkylating agents is known to correlate with the induction of at least two enzymes that repair a wide range of methylation lesions in DNA (Karran et al. 1982).

AlkA mutants of E. coli are sensitive to the toxicity of alkylating agents and are deficient in one of the inducible enzymes, 3-methyladenine-DNA glycosylase II (Evensen & Seeberg, 1982). The alkA gene has been cloned and sequenced and shown to be the structural gene of the protein (Clarke et al. 1984; Nakabeppu et al. 1984). The enzyme excises 3-methyladenine and 3-methylguanine, which are toxic to the cell, and also the minor alkylated pyrimidines, O1-methylcytosine and O1- methylthymine (Karran et al. 1982; McCarthy et al. 1984). The second inducible enzyme, O1-methylguanine-DNA methyltransferase, repairs the mutagenic lesions O1-methylguanine and O1-methylthymine by direct demethylation. The methyl groups are transferred from these lesions on to one of the enzyme’s cysteine residues identified as cysteine 321 in the C-terminal half of the protein (Fig. 1) (Olsson & Lindahl, 1980; Demple ei al. 1985). The same enzyme also repairs methylphosphotriester lesions in the DNA backbone. The ‘S’, but not the ‘R’, stereoisomer of methylphosphotriesters is repaired by methyl transfer to a different cysteine residue in the N-terminal region of the protein (McCarthy & Lindahl, 1985; Margison et al. 1985; Hamblin & Potter, 1985; Weinfeld et al. 1985). The location of this acceptor cysteine has yet to be determined. Cysteine 69 may be the second acceptor cysteine (Fig. 1) because it is within a protein sequence similar to the C-terminal active site. In accepting a methyl group from one O1-methylguanine or O1-methylthymine lesion and one methylphosphotriester lesion the methyltransferase is inactivated as a DNA repair enzyme.

Fig. 1.

Diagrammatic representation of the 39xlO1Mr (39 K) Ada protein, that is, the O1-methylguanine (O1MeG)-DNA methyltransferase. The putative position of the N-terminal active cysteine is within a similar amino sequence to the C-terminal active cysteine.

Fig. 1.

Diagrammatic representation of the 39xlO1Mr (39 K) Ada protein, that is, the O1-methylguanine (O1MeG)-DNA methyltransferase. The putative position of the N-terminal active cysteine is within a similar amino sequence to the C-terminal active cysteine.

The 39 X 101 Mr methyltransferase is very sensitive to proteolysis on cell lysis (Teo et al. 1984). As a result of this, the enzyme was originally purified as a 19 X 101Mr C- terminal fragment, which can repair O1-methylguanine and O1-methylthymine but not methylphos photriesters (see Fig. 1) (Demple et al. 1982). The protease responsible has not been identified, but resembles an activity that cleaves the UvrB protein in over-producing cells (Teo, 1986; Arikan et al. 1986).

The 39x 101Mr protein was shown, by immunological cross-reactivity, and DNA and protein sequencing, to be the product of the ada gene (Teo et al. 1984). Ada mutants were first characterized as regulatory mutants of the adaptive response as they lack both methyltransferase and glycosylase activities (Lindahl et al. 1983). The cloned ada gene product was found to stimulate in vivo expression of both its own gene and that of alkA, that is, it acts as a positive regulator of the adaptive response (Sedgwick, 1983; Demple et al. 1985; LeMotte & Walker, 1985). The Ada protein has, therefore, both regulatory and DNA repair activities, and in this respect is comparable with the RecA protein.

Two additional genes, alkB and aidB, have also been associated with the adaptive response. Expression of aidB is regulated by the Ada protein whereas alkB forms a small operon with ada. AlkB mutants are sensitive to alkylating agents whereas AidB mutants are resistant (Kataoka & Sekiguchi, 1985; Volkert & Ngyuen, 1984). The functions of the gene products are unknown.

We have recently been engaged in efforts to identify the inducing signal of the adaptive response produced in cells exposed to methylating agents, and to determine the role of the ada gene product in this regulatory mechanism.

Self-methylation of the Ada protein or its proteolytic cleavage have been proposed as possible mechanisms of its activation as a positive regulator of the adaptive response. The effects of addition of these various forms of the Ada protein on the expression of the ada and alkA genes were therefore examined in vitro using the techniques of DNA-directed protein synthesis and run-off transcription (Teo et al. 1986).

The plasmid pCS68 encodes three polypeptides, the products of the amp, tet and ada genes (Teo et al. 1984). The major 1S-labelled polypeptide synthesized in the Zubay system (Zubay, 1973) directed by pCS68 was the 31X 101Mr β -lactamase, the product of the amp gene. Little or no synthesis of the 36x 101Mr Tet protein or the 39X101Mr Ada protein was observed. Various forms of the Ada protein were added to this Zubay system in attempts to stimulate expression of the ada gene. Addition of the 39X101Mr Ada protein or its 19X101Mr C-terminal fragment did not induce synthesis of new Ada protein. However, the 39x 101Mr Ada protein, self-methylated by preincubation with MNU-treated DNA, was an efficient activator of ada gene expression (Teo et al. 1986). The methylated 19xl01Mr fragment did not induce expression.

During repair of methylated DNA, the Ada protein can be self-methylated at two active cysteines by accepting methyl groups from O1-methylguanine and methylphosphotriesters (see Fig. 1). To determine whether Ada protein methylated at only one of these sites could activate ada gene expression, the Ada protein was incubated with substrates containing only one of these sources of methyl groups. The substrate containing only O1-methylguanine was a double-stranded symmetrical oligonucleotide containing two O1-methylguanine • thymine base pairs. The substrate containing methylphosphotriesters but no O1-methylguanine was poly(dA) • poly(dT) methylated in only one of the strands. Methylated poly(dT) contains some O1- methylthymine but this was removed by mild acidic hydrolysis. The Ada protein singly methylated by repair of O1-methylguanine did not stimulate new synthesis of Ada protein in the pCS68-directed Zubay system. However, Ada protein singly methylated by repair of methylphosphotriesters was an efficient activator of ada gene expression. Transfer of a methyl group from a methylphosphotriester lesion in DNA on to a cysteine residue in the N-terminal region of the Ada protein is therefore the mechanism of activation of Ada protein as a positive regulator of its own expression.

In vivo observations made by LeMotte & Walker (1985) and Nakabeppu et al. (1986) have suggested that expression of the ada and alkA genes is regulated at the transcriptional level. In our experiments, run-off transcription of fragments of the ada and alkA genes, carrying the promoter regions and part of the structural genes, was not observed in the absence of added Ada protein. Addition of unmethylated protein resulted in a small increase in transcription, but this was increased approximately 100-fold by addition of methylated protein. The sizes of the transcripts agreed with those expected from in vivo determinations of the transcriptional start sites (Nakabeppu & Sekiguchi, 1986). The ability of unmethylated Ada protein to act as a weak activator accounts for the low levels of methyltransferase and glycosylase in uninduced cells, and for the higher levels of glycosylase observed in cells containing the ada gene on a multicopy plasmid (Sedgwick, 1983). Selfmethylation of the Ada protein therefore converts it to an efficient transcriptional activator of the ada and alkA genes (Teo et al. 1986).

Digestion of the Ada fragment with Hha\ in the promoter region upstream from the putative RNA polymerase binding site (see Fig. 3) resulted in a loss of the stimulatory effect of methylated Ada protein on run-off transcription. The methylated Ada protein may therefore be binding to the ada promoter in this region. Delayed migration on a polyacrylamide gel of an ada promoter fragment after incubation with methylated Ada protein also indicated that the protein was binding specifically to the ada promotor region. The ada plasmid pCS42 (Sedgwick, 1983) was cut with Awal andEcoRI into eight fragments, which were 1P-end-labelled and incubated with methylated Ada protein at a molar excess of zero to 1000-fold. Incubation with high concentrations of protein resulted in a slight but significant delayed migration on a polyacrylamide gel of the 276 base-pair fragment that contained the ada promoter region (Fig. 2). A shift to form a distinct new band of apparently higher molecular weight, as observed in similar experiments on other DNA binding proteins (Garner & Revzin, 1981; Hendrickson & Schleif, 1984) did not occur, possibly because of instability of the complex during migration through the polyacrylamide gel. The delayed migration of only the 276 base-pair fragment indicated that Ada protein was binding specifically to the ada promoter region.

Fig. 2.

Electrophoretic migration of an ada promoter DNA fragment was specifically delayed after incubation with methylated Ada protein. Ada protein was self-methylated by repair of MNU-treated DNA. An Aval/EctAA digest of the ada-carrying plasmid pCS42 (Sedgwick, 1983) was 1P-end-labelled, incubated with methylated Ada protein at various molar excesses for 30 min at 20 °C, and then analysed on a 5 % polyacrylamide gel. The smallest fragment of 276 base-pairs (bp) contains the ada promoter region and 76 bp of the structural gene. The molar excesses of methylated Ada protein were: lane 1, no added protein; lane 2, 90-fold; lane 3, 370-fold; lane 4, 1750-fold.

Fig. 2.

Electrophoretic migration of an ada promoter DNA fragment was specifically delayed after incubation with methylated Ada protein. Ada protein was self-methylated by repair of MNU-treated DNA. An Aval/EctAA digest of the ada-carrying plasmid pCS42 (Sedgwick, 1983) was 1P-end-labelled, incubated with methylated Ada protein at various molar excesses for 30 min at 20 °C, and then analysed on a 5 % polyacrylamide gel. The smallest fragment of 276 base-pairs (bp) contains the ada promoter region and 76 bp of the structural gene. The molar excesses of methylated Ada protein were: lane 1, no added protein; lane 2, 90-fold; lane 3, 370-fold; lane 4, 1750-fold.

Fig. 3.

DNA sequences of the promoters of the ada and alkA genes (upper and lower sequences, respectively) (Demple et al. 1985; Nakabeppu et al. 1984). The horizontal arrows indicate a region of dyad symmetry. The sequences protected by methylated protein from DNase I digestion are indicated. The broken lines indicate less well protected regions. The sequences common to the protected regions of both genes and the putative ribosome binding sites are boxed. The possible ribosomal binding sites of the alkA gene and the transcriptional start sites are from Nakabeppu & Sekiguchi (1986).

Fig. 3.

DNA sequences of the promoters of the ada and alkA genes (upper and lower sequences, respectively) (Demple et al. 1985; Nakabeppu et al. 1984). The horizontal arrows indicate a region of dyad symmetry. The sequences protected by methylated protein from DNase I digestion are indicated. The broken lines indicate less well protected regions. The sequences common to the protected regions of both genes and the putative ribosome binding sites are boxed. The possible ribosomal binding sites of the alkA gene and the transcriptional start sites are from Nakabeppu & Sekiguchi (1986).

The binding site was defined more precisely by DNase I footprinting experiments (Galas & Schmitz, 1978). End-labelled DNA fragments of the ada and alkA genes carrying the promoter regions and part of the structural genes were incubated with a 50-fold molar excess of methylated Ada protein and digested to a limited extent with DNase I. When the digests were examined on a denaturing polyacrylamide gel, the methylated Ada protein was observed to protect certain regions of the ada and alkA promoters (Teo et al. 1986; see Figs 3 and 4). The unmethylated protein did not protect these regions from DNase I digestion. The poor ability of the unmethylated protein to bind is in agreement with it being a weak activator of transcription. Selfmethylation of the Ada protein therefore enables it to bind specifically and efficiently to the ada and alkA promoters.

Fig. 4.

Protection of the alkA promoter against DNase I by methylated forms of the Ada protein. Ada protein (5pmol) was incubated with various substrates and then added to OTpmol of a 267 bp Accl-Mlul fragment of the alkA gene labelled with 3ZP at the 3’ end of the Accl site. The complexes were partially digested with DNase I and analysed on a denaturing 8% polyacrylamide gel. The DNA substrates were: lane 1, Micrococcus luteus DNA; lane 2, MNU-treated Micrococcus luteus DNA; lane 3, a mixture of the substrates in lanes 4 and 5; lane 4, poly(dA) • poly(dT) containing a MNU-treated poly(dA) strand; lane 5, a double-stranded oligonucleotide containing O-methyl- guanine. The mixtures were digested with various concentrations of DNase I to obtain similar levels of digestion of non-protected DNA. Lane 1, l ng µ1-1 for 15 s (a) and 30 s (b). Lanes 2, 3 and 4, 5ng uU1 for 10 s (a) and 30 s (b). Lane 5, 5ng µgl-1 for 30 s (a) and 60 s (b). Lane G is the Maxam-Gilbert G sequencing reaction of the same fragment. The DNA sequence is taken from Nakabeppu et al. (1984). The brackets indicate the promoter region protected by methylated Ada protein from DNase I digestion.

Fig. 4.

Protection of the alkA promoter against DNase I by methylated forms of the Ada protein. Ada protein (5pmol) was incubated with various substrates and then added to OTpmol of a 267 bp Accl-Mlul fragment of the alkA gene labelled with 3ZP at the 3’ end of the Accl site. The complexes were partially digested with DNase I and analysed on a denaturing 8% polyacrylamide gel. The DNA substrates were: lane 1, Micrococcus luteus DNA; lane 2, MNU-treated Micrococcus luteus DNA; lane 3, a mixture of the substrates in lanes 4 and 5; lane 4, poly(dA) • poly(dT) containing a MNU-treated poly(dA) strand; lane 5, a double-stranded oligonucleotide containing O-methyl- guanine. The mixtures were digested with various concentrations of DNase I to obtain similar levels of digestion of non-protected DNA. Lane 1, l ng µ1-1 for 15 s (a) and 30 s (b). Lanes 2, 3 and 4, 5ng uU1 for 10 s (a) and 30 s (b). Lane 5, 5ng µgl-1 for 30 s (a) and 60 s (b). Lane G is the Maxam-Gilbert G sequencing reaction of the same fragment. The DNA sequence is taken from Nakabeppu et al. (1984). The brackets indicate the promoter region protected by methylated Ada protein from DNase I digestion.

The Ada protein singly methylated by repair of methylphosphotriesters, but not O1-methylguanine, was also found to bind specifically to the same region of the ada promoter, which agrees with its ability to stimulate ada gene expression in the DNA- directed protein synthesis experiments described above (Teo et al. 1986). This singly methylated protein also protected the alkA promoter against DNase I digestion, however, the effect was only partial (Fig. 4), suggesting that it binds more readily to the ada than to the alkA promoter. This would agree with data indicating that the methyltransferase is induced to a greater extent than the glycosylase (Lindahl et al. 1983).

The sequence protected in the ada promoter is in the region of the HhaI site, in the centre of a region of dyad symmetry and immediately upstream from the putative RNA polymerase binding site (Fig. 3). The sequence protected in the alkA gene actually overlaps the proposed RNA polymerase binding site (Nakabeppu & Sekiguchi, 1986). Ada protein bound in these regions may therefore facilitate binding of RNA polymerase and in this way stimulate initiation of transcription.

The regions of the ada and alkA promoters protected by the methylated Ada protein have a long sequence in common, A-A-A-N-N-A-A-A-G-C-G-C-A, now referred to as the Ada box. This sequence is not present in any other E. coli promoters that are in the data banks (M. Ginsburg, personal communication). The only other gene known to be positively regulated by ada is the aidB gene, but the DNA sequence of this gene is not yet available.

Methylation of a specific cysteine in the N-terminal region of the Ada protein may produce a conformational change that enables the protein to bind specifically to the Ada box. This mechanism of activation is comparable with that of the cyclic AMPreceptor protein, which undergoes a conformational change on binding cyclic AMP, enabling it to bind to the promoters of catabolite-sensitive operons immediately upstream or overlapping their RNA polymerase binding sites (reviewed by de Crombrugghe et al. 1984; Raibaud & Schwartz, 1984). This regulatory mechanism is, however, quite different from that of the SOS response and the heat-shock response, which are also induced by stressful environments. The RecA protein is activated as a protease that destroys the lex A and other repressors of the SOS genes (Little & Mount, 1982; Walker, 1985). The regulatory gene of the heat-shock response encodes an alternative sigma factor of RNA polymerase, and in this way stimulates transcription of the heat-shock genes (Grossman et al. 1984; Landick et al. 1984).

Methylation of the Ada protein is the first example of covalent modification leading to activation of a regulatory protein (Teo et al. 1986). The protein is therefore also apparently irreversibly activated. Proteolytic cleavage of the Ada protein, which occurs rapidly on cell lysis, has not been reproducibly demonstrated in vivo, but it could possibly be of importance in destroying the activated Ada protein, and therefore in switching off the adaptive response. Preliminary evidence suggests that the 20X101 and 19X101Mr proteolytic fragments can still repair methylphosphotriesters and O1-methylguanine, respectively. Although the self-methylated N-terminal fragment was able to bind to the ada promoter, as determined by DNase I footprinting experiments, it was unable to stimulate run-off transcription of the ada gene. This fragment must, therefore, contain the DNA binding domain that recognizes the Ada box, but the Ada protein needs to be intact in order to enhance initiation of transcription.

Methylphosphotriesters in DNA are not known to have a toxic or mutagenic effect on the cells. DNA polymerase I is able to synthesize past these lesions although the rate of replication is reduced (Miller et al. 1982). The only known importance of actively repairing methylphosphotriesters in E. coli therefore appears to be the induction of the adaptive response.

The recent work reported in this review was performed at the Imperial Cancer Research Fund in collaboration with Drs I. Teo, M. Kilpatrick, T. McCarthy and T. Lindahl, and is described in greater detail by Teo et al. (1986).

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