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Oncogene (2002) 21, 8886 – 8894
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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
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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
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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)
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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
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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
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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).
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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
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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. The unstable glycosyl bond
of 3-meA, 3-meG, O2-meT or O2-meC in DNA is
hydrolytically cleaved by the AlkA DNA glycosylase,
but the more unusual strategies of direct demethylation
by the Ada DNA methyltransferase and the AlkB
DNA dioxygenase are required to repair more
chemically stable adducts. Ada transfers the methyl
groups from the methylated oxygens of O6-meG and
O4-meT in DNA on to itself, whereas AlkB oxidises
and labilizes the methyl groups in 1-meA and 3-meC.
O6-meG-DNA methyltransferases and 3-meA-DNA
glycosylases from human cells have been intensively
studied, and human AlkB is under investigation. These
human DNA repair enzymes may suppress oncogenic
transformation by endogenous and environmental
alkylating agents. On the other hand, they may have
the unfortunate effect of conveying cellular resistance
to many prospective anti-cancer drugs.
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