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ª Oncogene (2002) 21, 8886 – 8894 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Recent progress on the Ada response for inducible repair of DNA alkylation damage Barbara Sedgwick1 and Tomas Lindahl*,1 1 Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK Oncogene (2002) 21, 8886 – 8894. doi:10.1038/sj.onc. 1205998 Keywords: DNA repair; O6-methylguanine; DNA glycosylases Introduction Methylating agents comprise a major class of DNA damaging compounds that occur both endogenously and in the environment. They are extremely cytotoxic and frequently also mutagenic, and are employed for chemotherapy of certain cancers. All organisms have multiple DNA repair strategies to counteract the effects of DNA alkylation. To defend against fluctuating levels of environmental alkylating agents, many bacteria mount an inducible response that enhances cellular resistance to these same agents. This adaptive response has been most extensively studied in E. coli, in which induced alkylation resistance results from increased expression of four genes, ada, alkB, alkA and aidB. The Ada, AlkA and AlkB proteins protect against alkylation by repair of methylated bases in DNA using different mechanisms, whereas AidB may directly destroy certain alkylating agents. These DNA repair activities are conserved from bacteria to man. The Ada protein also serves as the positive regulator of ‘the adaptive response to alkylation damage’, which is more concisely named ‘the Ada response’ by analogy with the SOS, SoxRS and OxyR responses. Various aspects of the Ada response have been the subject of previous detailed reviews (Friedberg et al., 1995; Landini and Volkert, 2000; Lindahl et al., 1988; Samson, 1992; Sedgwick and Vaughan, 1991; Seeberg and Berdal, 1999). Here, we describe the properties of the inducible proteins with particular emphasis on recent findings, such as those from genome sequencing, protein structural studies, analysis of substrate specificities, and the newly discovered function of the AlkB protein. *Correspondence: T Lindahl; E-mail: [email protected] Methylating agents Methylating agents can alkylate DNA at many sites producing a wide variety of base lesions (Figure 1) and phosphotriesters. The relative proportions of the different base lesions depend on the nature of the alkylating agent, its reaction mechanism and the secondary structure of the DNA target. Of the commonly used laboratory alkylating agents, methyl methanesulphonate (MMS), dimethylsulphate (DMS) and methyl iodide (MeI) react via a SN2 mechanism and methylate DNA almost exclusively at nitrogen moieties in the purine and pyrimidine rings. In contrast, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and N-methylnitrosourea (MNU), which are SN1 agents, alkylate both nitrogens and oxygens in the DNA bases and also oxygens in the sugarphosphate backbone. The major lesions generated in double-stranded DNA are 7-methylguanine (7-meG), 3-methyladenine (3-meA) and O6-methylguanine (O6meG). In single-stranded DNA, SN2 agents also efficiently induce formation of 1-methyladenine (1meA) and 3-methylcytosine (3-meC) (Bodell and Singer, 1979). These lesions are less readily formed in duplex DNA because the modification sites are involved in base pairing, and are therefore shielded from alkylation. There are also some distinct minor sites of methylation. Several of the base modifications block DNA replication and are cytotoxic, including 3meA, 3-methylguanine (3-meG), 3-meC and probably also 1-meA. O6-meG and O4-methylthymine (O4-meT) mispair during DNA replication and are therefore mutagenic. It is fortuitous that the most abundantly formed lesion, 7-meG, is relatively innocuous (Singer and Grunberger, 1983). In a different type of DNA alkylation, environmental mutagens such as 1,2dimethylhydrazine, tert-butylhydroperoxide and diazoquinones generate methyl radicals that react with guanine residues in DNA to form miscoding 8methylguanine adducts (Hix et al., 1995). Conservation of the Ada response in many bacterial species suggests the presence of direct alkylating agents in their environment. Good candidate agents are those produced by microorganisms, but others may be formed by chemical reactions. Some Streptomyces sp. release alkylating antibiotics, such as streptozotocin (a derivative of MNU) and azaserine (O-diazoacyl-Lserine), into the soil creating an urgent need for an Repair of alkylated DNA B Sedgwick and T Lindahl 8887 It is of note that ada gene expression also increases in stationary phase to further the defence against alkylation; this enhanced expression is also dependent on RpoS (Taverna and Sedgwick, 1996). The preliminary studies on the function of AidB need to be extended, including more precise definition of its eukaryotic homologues. Figure 1 Sites of methylation on the DNA bases. Thick arrows indicate major sites of DNA methylation by SN1 agents. The curly arrow indicates an additional site alkylated by methyl radicals. Colours are used to indicate which damaged sites are repaired by particular DNA repair activities: blue by Ada; green by AlkA; red by AlkB; black, no known repair activity adaptive response in other microorganisms. Furthermore, certain algae and fungi growing in saline environments generate MeCl as a product of chloride detoxification (Sedgwick and Vaughan, 1991). MeCl is probably the most abundant methylating agent in our environment (Crutzen and Andreae, 1990). Chemically, direct acting alkylating agents may be formed by nitrosations, in slightly acidic conditions, of amides, amines, amino acids and peptides (Harrison et al., 1999; Sedgwick, 1997; Sedgwick and Vaughan, 1991). These reactions could possibly occur in decaying matter, in acidic soils or in putrid water. E. coli cells that are unable to repair O6-meG or O4meT residues have an increased frequency of spontaneous mutagenesis when subjected to starvation/ stationary conditions. Mutagenic alkylating agents must therefore arise in these cells, but their precise nature is unknown (Rebeck et al., 1989). S-Adenosylmethionine (SAM) weakly methylates DNA, but it acts by an SN2 mechanism and would not be expected to be an efficient mutagen. Thus, variation of the cellular SAM levels, over a 100-fold range, had no significant effect on spontaneous mutagenesis in E. coli (Posnick and Samson, 1999b). It is more likely that the major endogenous mutagen is a SN1 compound that alkylates oxygens in DNA. Bacterially catalyzed amine nitrosation increases in anaerobic and resting E. coli cells (Calmels et al., 1987; Kunisaki and Hayashi, 1979). A mutant deficient in this activity was less susceptible to spontaneous mutagenesis, suggesting that enzymatic nitrosation of amines or amides may be a source of endogenous mutagens (Harrison et al., 1999; Sedgwick, 1997; Taverna and Sedgwick, 1996). AidB protein shows some homology to the mammalian isovaleryl-coenzyme A dehydrogenases, and has been proposed to detoxify nitrosoguanidines (nitrosated amides) or their reaction intermediates (Landini et al., 1994). Expression of the aidB gene increases in anaerobic cells and is dependent on RpoS, the alternative sigma factor of RNA polymerase that controls expression of many genes in stationary phase (Volkert et al., 1994). A question of interest is whether elevation of AidB activity occurs in the same conditions as spontaneous mutagenesis and is required to destroy endogenously generated nitroso-compounds. Ada, an O6-meG-DNA methyltransferase and chemosensor for the adaptive response The multifunctional Ada protein repairs methylated bases and also regulates the adaptive response (Figure 2). Ada is composed of two major domains that can function independently in DNA repair reactions. The 19 kDa C-terminal domain (C-Ada19) directly demethylates the mutagenic bases, O6-meG and O4meT, and transfers the methyl groups on to its Cys-321 residue (Demple et al., 1985). Ada therefore precludes mutation induction by this rapid but suicidal action. The active site thiol of Cys-321 is buried in the 3-D structure of C-Ada19. The protein was therefore initially proposed to undergo a substantial conformational change on binding to its substrates to expose this active cysteine (Moore et al., 1994). An alternative model, however, used a predicted ‘helix – turn – helix – wing’ motif (HTH) to bind DNA and a nucleotide flipping mechanism to align the alkylated guanine with the catalytic cysteine (Vora et al., 1998). The more recent crystal structure of the human O6-meG-DNA Figure 2 Diagrammatic representation of the E. coli Ada response. The Ada protein is activated as a positive regulator by methylation of its Cys-38 residue in the amino-terminal half of the protein. This activation occurs by repair of methylphosphotriesters (PTE) in DNA or, less efficiently, by direct protein methylation. The activated Ada protein induces expression of several genes resulting in increased DNA repair and probable destruction of certain alkylating agents Oncogene Repair of alkylated DNA B Sedgwick and T Lindahl 8888 methyltransferase (AGT) also implicates a HTH motif in DNA binding and an ‘arginine finger’ to extrude the O6-alkG nucleotide from the duplex DNA. The extrahelical base can then reach the recessed AGT active site (Daniels and Tainer, 2000). These conserved motifs in the E. coli C-Ada19 domain are shown in Figure 3. A nucleotide flipping mechanism has been documented for several other DNA methyltransferases and DNA glycosylases (Cheng and Roberts, 2001). The Ada protein also demethylates Sp-diastereoisomers of methylphosphotriesters in DNA by methyl transfer on to the Cys-38 residue in the 20 kDa Nterminal domain (N-Ada20). This active cysteine was previously considered to be Cys-69 (Sedgwick et al., 1988), but has now been more accurately identified as Cys-38 in the recently solved 3-D structure of the protein (GL Verdine et al., submitted). N-Ada20 contains one Zn2+ tightly co-ordinated by 4 cysteine residues, Cys-38, Cys-42, Cys-69 and Cys-72 (Figure 3), which are all essential for biological activity. The bound metal is required for correct protein folding, and also activates the nucleophilicity of the methyl accepting Cys-38 residue. Methylation of Cys-38 does not completely abolish Zn2+ binding (Myers and Verdine, 1994). N-Ada20 has been a prototype for several other proteins that catalyze methyl group transfer reactions using Zn2+ co-ordinated thiols as nucleophiles, for example, enzymes involved in methionine synthesis and others in methanogenesis (Matthews and Goulding, 1997). Since C-Ada19 does not appear to contain a zinc residue to demethylate DNA by metallo-activated methyl transfer, the previously observed amino acid homology around residues Cys69 and Cys-321 may be coincidental. Self-methylation at Cys-38 converts Ada into a transcriptional activator and dramatically increases its sequence specific binding to the promoters of the adaalkB operon and the alkA and aidB genes (Landini and Volkert, 2000; Sakumi and Sekiguchi, 1989; Teo et al., 1986) (Figure 2). The transferred methyl group does not contact the promoter DNA, but may induce a conformational change in the protein that creates a new high affinity DNA binding face (Lin et al., 2001). A possible HTH motif and a basic segment of the protein may have roles in specific DNA binding (Sakashita et al., 1995) (Figure 3). Self-methylated Ada binds to the ada and aidB promoters immediately upstream of the RNA polymerase binding sites, and CAda19 may be required for direct interaction with RNA polymerase. In contrast, the self-methylated NAda20 is sufficient to activate the alkA gene (Akimaru et al., 1990). The mechanisms by which self-methylated Ada interacts with the promoters of the Ada regulon and RNA polymerase have been reviewed in detail (Landini and Volkert, 2000). E. coli has a second O6-meG-DNA methyltransferase (Ogt) and a second 3-meA-DNA glycosylase (Tag) that are expressed constitutively, and will repair some O6meG and 3-meA lesions in DNA during the vulnerable period in which the adaptive response is being induced (Samson, 1992). Ingeniously, methylphosphotriesters, a relatively innocuous type of DNA damage, that are not repaired by Ogt or any other known constitutive activity, are used as the inducing signal for the adaptive response. Repair of one of the two stereoisomers of methylphosphotriesters therefore serves solely as a sensor for changing levels of DNA alkylation damage in bacteria (Lindahl et al., 1988). Figure 3 Alignment of the E. coli Ada and B. subtilis AdaA proteins with other putative bacterial homologues. Protein accession references are: Paracoccus pantotrophus, AAK61311; Mesorhizobium loti, NP_103342; E. coli, NP_416717; B. subtilis, NP_388062; Lactococcus lactis, NC_002662. Identical and conserved residues are highlighted in yellow, and similar residues in grey. A hinge region between the two major domains of the E. coli protein is indicated (Sedgwick et al., 1988). Red stars indicate the cysteine residues that ligand Zn2+ (Cys-38, Cys-42, Cys-69 and Cys-72 in the E. coli protein). ‘CH3’?points to the methyl accepting cysteines, indicated in red for methylphosphotriester repair and green for O6-meG repair (corresponding to Cys-38 and Cys-321 in the E. coli protein). Putative HTH DNA binding motifs occur in both domains. Within the proposed HTH of the C-terminal domain is a conserved ‘arginine finger’ which is implicated in nucleotide flipping. An invariant Asn-hinge couples the recognition helix (second helix) to the active site cysteine in this domain (Daniels and Tainer, 2000) Oncogene Repair of alkylated DNA B Sedgwick and T Lindahl 8889 Eukaryotic organisms do not appear to have a DNA methyltransferase that repairs methylphosphotriesters. Presumably this is not required as eukaryotes do not have an inducible response equivalent to that in bacteria. Cys-38 of the E. coli Ada protein can be directly methylated by MeI (GL Verdine et al., submitted). Indeed, direct methylation of this cysteine by SN2 agents may be an alternative but less effective mode of Ada activation as a positive gene regulator (Figure 2) (Takahashi and Kawazoe, 1987). SN1 agents induce the response by efficiently forming methylphosphotriesters in DNA, whereas SN2 methylating agents may cause induction by inefficiently generating a few phosphotriesters in a large chromosomal DNA target and then, as the cellular level of Ada increases, by direct Ada methylation (Vaughan et al., 1993) (Figure 2). The Ada response conveys a defence against both types of agents, against killing by SN2 agents and against both mutagenicity and toxicity of SN1 agents. It therefore seems logical that both types of agents should have a means of inducing the response. Once all the methylphosphotriesters in DNA have been repaired, unmethylated Ada protein will accumulate in the cell. High concentrations of unmethylated protein (4200 molecules per cell) inhibit ada gene activation by the methylated form, and will consequently switch off the adaptive response in the absence of further damage (Saget and Walker, 1994). AlkB, a 1-meA/3-meC DNA dioxygenase Independently of the discovery of the Ada response, E. coli alkA and alkB mutants were isolated in screens for MMS sensitive mutants (Clarke et al., 1984; Kataoka et al., 1983; Yamamoto et al., 1978). Although the function of the AlkA protein was soon elucidated, the role of AlkB was only recently resolved (Dinglay et al., 2000; Falnes et al., 2002; Trewick et al., 2002). Evidence that AlkB acts alone came from its ability to confer MMS resistance to human cells (Chen et al., 1994). Also, the observation that AlkB repairs methylated single-stranded DNA in vivo predicted that 1-meA and 3-meC were its substrates (Dinglay et al., 2000). Despite these findings, many attempts to develop in vitro assays for AlkB were unsuccessful until clues of the unusual cofactor requirement were revealed (Aravind and Koonin, 2001). The substrates of AlkB have now been identified as 1-meA and 3-meC in DNA (Trewick et al., 2002). These lesions (Figure 1) are predominantly generated in single-stranded DNA by SN2 methylating agents, MMS, DMS or MeI, and to a much lesser degree by SN1 agents, MNNG and MNU (Bodell and Singer, 1979; Singer and Grunberger, 1983). The repair of these modifications by AlkB explains the phenotypic characteristics of alkB mutants: their greater sensitivity to SN2 than to SN1 alkylating agents (Kataoka et al., 1983), their inability to reactivate single-stranded DNA phage treated with SN2, but not SN1, agents, and their very small defect in the reactivation of double-stranded DNA phage treated with SN2 agents (Dinglay et al., 2000; Kataoka et al., 1983). Although 1-meA and 3meC are generated mainly in single-stranded DNA, AlkB can repair these modifications not only in singlestrands, but also in double-stranded structures obtained by reannealing. The low level of MMS induced mutagenesis in E. coli alkB mutants suggests that unrepaired 1-meA and 3-meC have a low capacity for mispairing during DNA replication, and instead block replication (Dinglay et al., 2000). MMS treated poly(dC) and poly(dA) contain lesions that block DNA synthesis in vitro, and these are presumably 3-meC in poly(dC), and both 1-meA and 3-meA in poly(dA) (Boiteux and Laval, 1982; Larson et al., 1985). AlkB was recently shown to be an a-ketoglutarateFe(II)-dependent dioxygenase, and repairs 1-meA and 3-meC in DNA by oxidative demethylation. Theoretical protein fold recognition predicted that AlkB was a member of this superfamily of enzymes (Aravind and Koonin, 2001) (Figure 4), and this was verified by biochemical analyses (Falnes et al., 2002; Trewick et al., 2002). These non-heme iron enzymes require Fe(II) as a cofactor, and a-ketoglutarate and O2 as cosubstrates. They catalyze the hydroxylation of an unactivated C-H bond in the substrate coupled to the oxidative decarboxylation of a-ketoglutarate, from which the products are succinate and CO2. One atom of oxygen from O2 is incorporated as a hydroxyl in the substrate and the other in the carboxylate group of succinate (Figure 5a) (for reviews see Prescott and Lloyd, 2000; Ryle and Hausinger, 2002; Schofield and Zhang, 1999). Using this mechanism, AlkB reverts 1meA and 3-meC in DNA directly to adenine and Figure 4 Topological diagrams for three members of the a-ketoglutarate-Fe(II) dioxygenase family. The diagrams are based on the experimentally determined structure for E. nidulans isopenicillin-N-synthase and structural models of prolyl-4-hydroxylase and AlkB. Conserved amino acid residues of the postulated active site and Fe(II) binding site are shown (reproduced with permission from Aravind and Koonin, 2001). The C-terminal third of the AlkB protein contains sequences that are not present in other family members, and may be required for DNA binding and substrate recognition Oncogene Repair of alkylated DNA B Sedgwick and T Lindahl 8890 Figure 5 (a) Basic enzymatic reaction catalyzed by a-ketoglutarate-Fe(II)-dependent dioxygenases. Iron-oxo intermediates oxidize inert substrates coupled to the oxidative decarboxylation of a-ketoglutarate to succinate. (b) Schematic representation of the repair of 1-meA and 3-meC residues in DNA by AlkB. Oxidation of 1-meA and 3-meC by AlkB requires Fe(II) as a cofactor, O2 and a-ketoglutarate as co-substrates, and generates CO2 and succinate. Oxidized methyl groups are released as formaldehyde resulting in direct reversal of the lesions to the unmodified base residues. Proposed intermediates are shown. Note that the alkylation positions in 1-meA and 3-meC are equivalent, although these sites in the pyrimidine rings of A and C are numbered differently by standard nomenclature cytosine with the release of the oxidized methyl group as formaldehyde (Trewick et al., 2002). The intermediate products are probably 1hydroxymethyladenine and 3-hydroxymethylcytosine that are expected to be unstable and decompose to release formaldehyde (Figure 5b). Enzymes in the superfamily of a-ketoglutarate-Fe(II)-dependent dioxygenases catalyze a variety of reactions including hydroxylations, desaturations and oxidative ring closures. For comparison with AlkB, another enzyme that oxidizes methyl groups is thymine hydroxylase. This dioxygenase, involved in the pyrimidine salvage pathway of the fungi Neurospora crassa and Rhodotorula glutinis, oxidizes the methyl group of free thymine, and by three successive oxidations forms 5-hydroxymethyluracil, 5-formyluracil and 5-carboxyuracil. To finally generate uracil, the C-C bond of 5-carboxyuracil is cleaved by a carboxylase. In vitro, thymine hydroxylase can also demethylate 1-methylthymine initially forming 1-hydroxymethylthymine which decomposes to thymine and formaldehyde (Thornburg et al., 1993). This latter reaction appears to be similar to the demethylation of 1-meA and 3-meC by AlkB. Furthermore, these oxidation reactions catalyzed by AlkB and thymine hydroxylase are similar to those of cytochrome P450 which demethylates N-methylamines (Yang and Smith, 1996) and amine oxidases that Oncogene deaminate methylamine, both releasing formaldehyde (Lizcano et al., 2000). AlkB repairs lesions generated in single-stranded DNA, and may act at DNA replication forks and transcription bubbles. More efficient binding of AlkB to single- than to double-stranded DNA may direct AlkB to these regions (Dinglay et al., 2000). The hypothesis that AlkB acts at DNA replication forks and actively transcribed regions explains why rapidly growing E. coli alkB cells are more sensitive to MMS than those in stationary phase (Dinglay et al., 2000). An association of AlkB with DNA synthesis is also seen in Caulobacter crescentus in which the alkB gene is co-expressed with activities required for DNA replication. In C. crescentus, neither AlkB nor Ada are induced by MMS, thus an Ada response is not apparent in this bacterium (see below Figure 7) (Colombi and Gomes, 1997). Direct reversal by AlkB of 1-meA and 3-meC to adenine and cytosine in DNA indicates an accurate mode of DNA repair. Other precise mechanisms of direct reversal of DNA damage are those catalyzed by suicidal O6-methylguanine-DNA methyltransferases, light requiring photolyases that monomerize cyclobutyl pyrimidine dimers (Sancar, 1996) and the B. subtilis spore SP lyase which reverses the unique spore photoproduct (5-thyminyl-5,6-dihydrothymine dimer) to two thymines. Similarly to AlkB, SP lyase was discovered 20 years ago but has been only recently biochemically characterized (Rebeil and Nicholson, 2001). This enzyme is a member of the ‘radical SAM’ superfamily that use an (Fe-S) centre and S-adenosylmethionine to generate adenosyl radicals to effect catalysis. Thus, both AlkB and SP lyase use metal catalysis to carry out difficult chemical reactions in DNA repair, free radical intermediates being required to attack the stable lesions. Site specific mutagenesis and crystal structure determinations have identified a conserved motif of three non-adjacent amino acids, His-X-Asp/Glu-XnHis as part of the Fe(II) ligand site in a-ketoglutaratedependent dioxygenases. This triad is also conserved in the E. coli AlkB protein and several of its putative homologues (Figures 4 and 6). An alkB mutant of C. crescentus is sensitive to MMS (Colombi and Gomes, 1997), verifying that the C. crescentus sequence is a true homologue. Curiously, the second His of the proposed iron ligand site in this sequence is replaced by Ser. This is also apparent in putative Mesorhizobium loti (Figure 6) and Sinorhizobium meliloti homologues (data not shown). A different His residue closer to the C-terminus may provide the third iron coordinating ligand in these proteins. Crystal structures of a-ketoglutarate-dependent dioxygenases suggest that a-ketoglutarate forms an electrostatic interaction with a conserved Arg residue (Zhang et al., 2000). Multiple sequence alignments of these enzymes with AlkB (Aravind and Koonin, 2001) indicate that a conserved Arg towards the C-terminus of the AlkB homologues may be involved in aketoglutarate interactions (Figure 6). Repair of alkylated DNA B Sedgwick and T Lindahl 8891 E. coli has constitutive and inducible O6-methylguanine-DNA methyltransferases (Ogt and Ada) and 3-methyladenine-DNA glycosylases (Tag and AlkA) (Lindahl et al., 1988; Seeberg and Berdal, 1999). The question of whether E. coli also has a constitutive activity that repairs 1-meA and 3-meC is unresolved. The extreme sensitivity of AlkB mutants to alkylation in single-stranded DNA (Dinglay et al., 2000) implies that a constitutive activity may not exist. Moreover, database searches of the E. coli genome have not revealed any significant candidate homologue for such an activity (P Bates and B Sedgwick, unpublished data). AlkB activity involves iron-oxo intermediates which may themselves be a threat to the cell due to oxidative DNA and protein damage. Consequently, it may be an evolutionary advantage to have high cellular levels of AlkB only when it is absolutely essential for survival, as occurs on exposure of E. coli to methylating agents or possibly during DNA replication in C. crescentus. The E. coli ada and alkB genes occur in a small operon regulated from the ada promoter. We can therefore predict that the AlkB protein will be present at a similar low level to the Ada protein in the uninduced cell, that is, approximately 2 to 4 molecules per cell (Potter et al., 1989; Rebeck et al., 1989; Vaughan et al., 1991). Although putative alkB sequence homologues are widespread from bacteria to humans, they are not universal, and are not found in the complete genome sequences of some microorganisms, for example, B. subtilis and Haemophilus influenzae, Archea or Saccharomyces cerevisiae. Curiously, three genes isolated from S. cerevisiae complement the sensitivity of an E. coli alkB, but not a recA, mutant to MMS. One of these genes encodes a protein kinase whereas the others may be membrane glycoproteins. The explanation for this complementation remains unclear (Wei et al., 1995). Several candidate human AlkB homologues are found in the databases (P Bates and B Sedgwick, unpublished data), and it must be ascertained which of these have roles in DNA repair. AlkA, a DNA glycosylase with broad substrate specificity 3-Methyladenine is an abundant alkylation lesion generated by both SN1 and SN2 alkylating agents. It is a strongly cytotoxic base because its methyl group protrudes into the minor groove of the DNA double helix, where it can block DNA replication and transcription. This lesion is repaired efficiently in all living cells. E. coli possesses a constitutively expressed 3-methyladenine-DNA glycosylase, the product of the tag gene, which appears highly specialized for the excision of 3-methyladenine (Lindahl et al., 1988; Seeberg and Berdal, 1999). The E. coli alkA gene encodes a second 3-methyladenine-DNA glycosylase, which is inducible as part of the Ada response. The AlkA enzyme has a much broader substrate specificity than Tag; it excises the minor lesion 3-methylguanine relatively efficiently, whereas this is a poor substrate for Tag. AlkA also catalyzes the release from DNA of the Figure 7 Functional domain fusions and diverse gene organisation in various bacterial species. Different coloured boxes represent different functional domains. Blue, AdaA; green, AdaB; yellow, AlkA; red, AlkB. The domains exist as separate proteins in B. subtilis, but are fused in different combinations in E. coli, C. crescentus and Streptomyces coelicolor. Major lesions repaired by the different domains are indicated below the E. coli proteins. Small cross-hatched boxes represent Ada regulated promoters. In C. crescentus, the alkB gene is co-expressed with genes involved in DNA replication Figure 6 Alignment of the E. coli and C. crescentus AlkB proteins with other putative bacterial homologues. Protein accession references are: Vibrio cholerae, NP_23345; Pseudomonas aeruginosa, NP_251996; Xanthomonas campestris, NP_636023; E. coli, NP_416716; Brucella melitensis, NP_541717; C. crescentus, NP_418829; Mesorhizobium loti, NC_002678. Identical and conserved residues are highlighted in yellow, and similar residues in grey. Red stars indicate the Fe(II) ligand triad, HisXAspXnHis. The green star indicates an arginine residue that is possibly involved in a-ketoglutarate binding Oncogene Repair of alkylated DNA B Sedgwick and T Lindahl 8892 minor products O2-methylcytosine and O2-methylthymine formed by SN1 agents, as well as the 8methylguanine lesion generated by methyl radicals (Gasparutto et al., 2002). Furthermore, the AlkA enzyme liberates altered adenines such as ethenoadenine generated as a consequence of exposure of DNA to lipid peroxidation products, hypoxanthine which is identical with hydrolytically deaminated adenine, and formyluracil and a ring fragmentation product of thymine generated by active oxygen (Privezentzev et al., 2000). These lesions are not generated by alkylating agents, so it is unclear why they should be repaired by one of the enzymes of the Ada response. However, AlkA is present in cells at a higher constitutive level than Ada and AlkB, and may be sufficient to deal with the small amounts of endogenously generated lesions. The identification of 3-meA as a cytotoxic rather than a mutagenic DNA lesion has been confirmed by studies using the minor groove-specific alkylating agent Me-lex, a methyl sulphonate ester attached to a dipeptide (Monti et al., 2002). This alkylating agent has a restricted target specificity; it forms 3-meA efficiently, but does not generate adducts in the major groove of DNA, and binds preferentially to A/T rich sequences. The glycosyl bond of 3-methyldeoxyadenosine moieties in DNA is relatively weak and has a half life of only 20 – 30 h at 378C and neutral pH. Whilst this rate of spontaneous hydrolytic cleavage is so slow that an active repair mechanism is required for removal of the toxic 3-meA residues, a 104-fold acceleration of the cleavage rate is sufficient to allow rapid DNA repair. This appears an easy task for enzymes, which are often required to speed up reaction rates by 106 – 108-fold. In consequence, several reaction mechanisms have evolved for DNA glycosylases to remove 3-MeA from DNA in different organisms, giving the impression that 3-methyladenine-DNA glycosylase sequences are poorly conserved. Thus, there is no obvious sequence homology between Tag, AlkA, and the human enzyme although this latter protein has a similarly structured active site to AlkA. An intriguing consequence of the broad substrate specificity of AlkA is that the enzyme can release the normal bases guanine and adenine from DNA at a very slow rate (Berdal et al., 1998). This is such a sluggish reaction that it is hardly relevant in vivo relative to the rate of spontaneous hydrolytic depurination. At very high cellular levels of AlkA protein this side reaction is troublesome, however, and may account for the surprisingly poor alkylation resistance of cells that greatly overexpress AlkA. By the same reasoning, it would make sense to keep AlkA levels relatively low in normal proliferating cells, and only induce the enzyme as part of the Ada response after a specific challenge by excessive DNA alkylation (Berdal et al., 1998; Posnick and Samson, 1999a). The 3-D structure of AlkA is available and has clarified the catalytic properties of the enzyme (Labahn et al., 1996; Yamagata et al., 1996). A comprehensive review (Hollis et al., 2000) describes the main structural features of AlkA. Briefly, the Oncogene enzyme is comprised of three sub-domains and, as found in several different DNA glycosylases, has a helix – hairpin – helix motif for interaction with damaged DNA positioned opposite a catalytic Asp residue in the active site. Similarly to most other enzymes in the DNA glycosylase family, AlkA introduces a bend in its DNA substrate, followed by flipping-out of the damaged deoxynucleoside residue which is rotated towards the active site. Whereas other DNA glycosylases with more precise and restricted substrate specificity tend to place the substrate residue in a deep and tightly fitting pocket, AlkA has a shallow groove or cleft, rich in aromatic and hydrophobic residues, which can accommodate the diverse substrates of the enzyme. The prime consideration, however, should be the efficient excision of 3methyladenine from DNA. In this regard, it is interesting that a 3-methyladenine-DNA glycosylase, induced in Helicobacter pylori on exposure to MNNG, has a more restricted substrate specificity similar to that of the E. coli Tag enzyme (O’Rourke et al., 2000). In spite of the detailed structural information on AlkA, it is still not entirely clear which features of the substrate are specifically recognized by the enzyme. One possibility is that the altered charge distribution in the key substrates, allowing for stronger interactions with electron-rich aromatic amino acid residues in the active site, is a relevant recognition factor (Labahn et al., 1996). An alternative model postulates that the decreased stability of the glycosyl bond in the modified deoxynucleoside residue is of crucial importance (Seeberg and Berdal, 1999). Adaptive responses and diverse domain fusions in different bacteria A variable capacity for an Ada response occurs among bacterial species. Such responses have been observed by induction of cellular resistance to alkylating agents, by enhanced DNA repair activities or by immunodetection of increased Ada protein levels. Conserved protein homologues are now also found in the sequenced genomes of many prokaryotes and eukaryotes. The bacterial Ada and AlkA proteins provide an excellent example of the evolutionary fusion of protein domains with related roles into ‘composite’ proteins. Component domains that exist as separate proteins in one species are fused in different combinations in others (Marcotte et al., 1999). The 39 kDa Ada protein of E. coli is composed of two domains that account for different DNA methyltransferase activities. Ada orthologues of similar molecular weight are induced in several other enterobacteria and Pseudomonas aeruginosa (Sedgwick and Vaughan, 1991), and database searches reveal homologues in other species, such as, Mesorhizobium and Paracoccus (Figure 3). In B. subtilis, the two functional domains exist as separate proteins, AdaA and AdaB, that demethylate methyl- Repair of alkylated DNA B Sedgwick and T Lindahl 8893 phosphotriesters and O6-methylguanine in DNA respectively (Figure 7). Induction of AdaA and AdaB proteins also occurs in B. cereus, but several other Bacillus species show a limited or undetectable inducible response (Morohoshi and Munakata, 1995). Nevertheless, putative AdaA homologues are found in the genomes of B. halodurans, B. anthracis and Lactococcus lactis (Figure 3). Intriguingly, a further mode of domain fusion, that of AdaA with AlkA, occurs in Mycobacterium tuberculosis, Streptomyces coelicolor, Xanthomonas campestris and Vibrio cholerae (Seeberg and Berdal, 1999; B Sedgwick, unpublished data) (Figure 7). Fusion of AdaA with AlkA in different bacterial species seems to be almost as common as AdaA with AdaB. Organization of the adaptive response genes also differs between different species (Figure 7). The ada and alkB genes form an operon in E. coli and alkA is located separately. In contrast, in B. subtililis, the adaA and adaB constitute an operon with the alkA in an adjacent position, and no alkB gene has been detected. The methylated AdaA protein of B. subtilis regulates the adaA – adaB operon and the divergent alkA gene by binding to a central region between their transcriptional start sites (Morohoshi et al., 1993). In M. tuberculosis and S. coelicolor, the adaA – alkA gene is adjacent to an adaB homologue. An inducible 17 kDa O6-methylguanine-DNA methyltransferase reported in V. cholerae (Bhasin and Ghosh, 1995) could be the AdaB protein. Conclusions All four of the major chemically methylated bases, and also four minor modifications, are repaired by the versatile inducible enzyme activities of the Ada response (Figures 1 and 2). It remains unknown whether two very minor lesions, 3-methylthymine and 1-methylguanine, are repaired at all, or whether they slowly accumulate as persistent lesions. A striking feature of the inducible activities is the diverse mechanisms that are required to remove the different alkylated bases from DNA. 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