journa l homepage: www.e lsev ier .com/ locate /dnarepai r
he ada operon of Mycobacterium tuberculosis encodes two DNAethyltransferases for inducible repair of DNA alkylation damage
ingyi Yanga,b,c, Randi M. Aamodta,c, Bjørn Dalhusa,b,c, Seetha Balasinghama,c, Ina Helleb,c,ernille Andersenb,c, Tone Tønjuma,c, Ingrun Alsetha,c, Torbjørn Rognesa,c,d, Magnar Bjøråsa,b,c,∗
Department of Microbiology, Oslo University Hospital, Rikshospitalet, PO Box 4950 Nydalen, NO-0424 Oslo, NorwayInstitute of Clinical Biochemistry, University of Oslo, Rikshospitalet, PO Box 4950 Nydalen, NO-0424 Oslo, NorwayCentre for Molecular Biology and Neuroscience, Oslo University Hospital, Rikshospitalet, PO Box 4950 Nydalen, NO-0424 Oslo, NorwayDepartment of Informatics, University of Oslo, PO Box 1080 Blindern, NO-0316 Oslo, Norway
r t i c l e i n f o
rticle history:eceived 2 December 2010eceived in revised form 1 March 2011ccepted 15 March 2011vailable online 12 May 2011
eywords:. tuberculosisNA damagelkylation
a b s t r a c t
The ada operon of Mycobacterium tuberculosis, which encodes a composite protein of AdaA and AlkAand a separate AdaB/Ogt protein, was characterized. M. tuberculosis treated with N-methyl-N′-nitro-N-nitrosoguanidine induced transcription of the adaA-alkA and adaB genes, suggesting that M. tuberculosismount an inducible response to methylating agents. Survival assays of the methyltransferase defectiveEscherichia coli mutant KT233 (ada ogt), showed that expression of the adaB gene rescued the alkyla-tion sensitivity. Further, adaB but not adaA-alkA complemented the hypermutator phenotype of KT233.Purified AdaA-AlkA and AdaB possessed methyltransferase activity. These data suggested that AdaB coun-teract the cytotoxic and mutagenic effect of O6-methylguanine, while AdaA-AlkA most likely transfersmethyl groups from innocuous methylphosphotriesters. AdaA-AlkA did not possess alkylbase DNA gly-
dalkAethyltransferaseNA glycosylase
cosylase activity nor rescue the alkylation sensitivity of the E. coli mutant BK2118 (tag alkA). We proposethat AdaA-AlkA is a positive regulator of the adaptive response in M. tuberculosis. It thus appears that theada operon of M. tuberculosis suppresses the mutagenic effect of alkylation but not the cytotoxic effectof lesions such as 3-methylpurines. Collectively, these data indicate that M. tuberculosis hypermutatorstrains with defective adaptive response genes might sustain robustness to cytotoxic alkylation DNAdamage and confer a selective advantage contributing to host adaptation.
Methylating agents comprise a major class of DNA damaginggents that are found both endogenously and in the environment.ells have evolved multiple DNA repair mechanisms to counter-ct the cytotoxic and mutagenic effect of methylation on DNA1]. 3-Methylpurines (3mA and 3mG) and 1-methyladenine (1mA)re major cytotoxic lesions repaired by the base excision repairathway (BER) and oxidative demethylation, respectively, while6-methylguanine (O6-mG) is a major mutagenic lesion removed
y methyltransferases (for review) [2,3].
Many bacteria counteract the deleterious effect of environmen-al exposure to alkylating agents by an inducible response termed
∗ Corresponding author at: Department of Microbiology, Oslo University Hospital,ikshospitalet, PO Box 4950 Nydalen, NO-0424 Oslo, Norway. Tel.: +47 23074060;
the adaptive response or Ada response [3]. In Escherichia coli fourgenes are induced by the adaptive response, ada, alkA, alkB and aidB,in which the methyltransferase Ada acts as a positive regulator ofthe operon. The 3mA DNA glycosylase AlkA is induced 10 fold whencells are exposed to sublethal doses of alkylating agents. A secondconstitutively expressed 3mA DNA glycosylase Tag is the majoralkylbase DNA glycosylase activity in E. coli under normal growthconditions. The AlkB protein is an Fe (II)-dependent dioxygenasethat repairs lesions such as 1mA and 3mC by oxidative demethyla-tion [4,5]. AidB is a DNA binding protein predicted to catalyze directrepair of alkylated DNA [3,6].
E. coli Ada is composed of two major domains: the N-terminalAdaA fold and the C-terminal AdaB fold. Methyl groups of the muta-genic and cytotoxic bases O6-mG and O4-methylthymine (O4-mT)are transferred to the Cys321 residue residing in the 19 kDa AdaBdomain [7]. The 20 kDa AdaA domain removes the methyl group of
innocuous methylphosphotriesters (MPT) in DNA by transfer to itsCys38 residue. The methylation of Cys38 converts the Ada proteininto a transcriptional activator with specific DNA binding affinityto genes containing the ada operator sequence in their promoters,
ncluding the ada gene itself. Unmethylated Ada protein inhibitsranscriptional activation mediated by the methylated form [8].onsequently, MPTs act as molecular sensors for fluctuating lev-ls of DNA alkylation in bacteria [9]. Additionally, E. coli harborssecond constitutively expressed O6-mG DNA methyltransferase,gt, which is homologous to the AdaB domain [10].
The adaptive response is conserved among many bacterialpecies, in which protein homologues have been identified fromequenced genomes. Notably, the domains of Ada and AlkA proteinsxist in diverse combinations in different prokaryotes. Further-ore, the genomic organization of genes involved in the adaptive
esponse differs between various species. In Mycobacterium tuber-ulosis, a domain fusion of AdaA with AlkA constitutes an operonith the ogt gene, here termed adaA-alkA and adaB, respectively.
earches in genome databases reveal that the AdaA-AlkA fusioneems to be as common as AdaA with AdaB in bacteria [3].
M. tuberculosis is probably the most widespread humanathogen and tuberculosis is the cause of more deaths in adultsorldwide than any other infectious disease, with the high-
st incidence found in developing countries. This pathogen ismember of the M. tuberculosis complex, which groups the
ovis BCG, Mycobacterium caprae, Mycobacterium pinnipedii andycobacterium microti. As a facultative intracellular pathogen, M.
uberculosis survives and replicates inside human macrophages.ccordingly, it is subjected to a hostile environment in whichlkylating stress and reactive oxygen- and nitrogen radicals are pro-uced that can induce deleterious effects, including DNA damage.revious studies have suggested that sequence variations in severaltrains of M. tuberculosis have led to transient mutator phenotypesnd host adaptation [4,11,12]. In this work we have characterizedhe ada operon of M. tuberculosis by biochemical analysis and func-ional complementation assays of E. coli mutants. Activity assaysith purified recombinant proteins demonstrated that both AdaB
nd AdaA-AlkA possess DNA methyltransferase activity, whereaso 3mA DNA glycosylase activity could be detected. Furthermore,daB suppressed the hypermutator phenotype of the E. coli adagt mutant while AdaA-AlkA showed no effect. These results sug-est that AdaB counteract the mutagenic effect of O6-mG, whereasdaA-AlkA possesses a methyltransferase activity that removesethyl groups from innocuous MPTs.
M. tuberculosis H37Rv was grown in Middlebrook 7H9 mediumith ADC and 0.05% Tween 80 at 37 ◦C while shaking until OD600as 0.5. The cultures were divided into two equal volumes, of which
ne of the cultures was treated with 3 �M N-methyl-N′-nitro-N-itrosoguanidine (MNNG) for 60 min. The total RNA was isolatedsing RNeasy spin columns (Qiagen) as described in Olsen et al.13].
.2. Quantitative RT-PCR
The cDNA was synthesized from total RNA using the High-apacity cDNA reverse transcription kit from Applied BiosystemsFoster City, CA) according to the manufacture’s protocol. The
uantitative PCR reaction was performed using the StepOnePlusTM
nstrument (Applied Biosystems, Foster City, CA) with the PowerYBR® Green PCR Master Mix kit (Applied Biosystems, Warrington,K). Each cDNA sample was analyzed in triplicate. The expression
10 (2011) 595–602
level of the housekeeping gene sigA was used as endogenous con-trol to normalize the relative expression level of target genes. Astandard curve of Ct value was generated with a serial dilution ofsigA cDNA to confirm that the real-time PCR reactions were run inthe linear range. Primer pairs (sequence details in Supplementarydata Table S1) for qPCR were designed with the software PrimerExpress provided by Applied Biosystems.
2.3. E. coli strains, plasmids and DNA constructs
E. coli ER2566 (New England Biolabs) was used for cloning andoverexpression of M. tuberculosis AdaA-AlkA and AdaB. The E. colistrains AB1157 (wild type), BK2118 (tag alkA) [14] and KT233 (adaogt) [15] were used in survival and mutagenesis experiments. TheadaA-alkA and adaB genes were amplified by PCR using M. tuber-culosis genomic DNA as template. In addition, the adaA and alkAdomains of the adaA-alkA gene were amplified separately. All PCRfragments were cloned into the BamHI and PstI sites of pUC18(New England Biolabs). The resulting plasmids were termed pUC-adaA, pUC-alkA, pUC-adaB and pUC-adaA-alkA. Next, the ORFs ofadaA-alkA and adaB were inserted into the BamHI and NdeI sitesof pET28b (Novagen), which were termed pET-adaA-alkA and pET-adaB. Primers used for cloning are listed in Supplementary dataTable S1.
2.4. Survival and mutagenesis
Overnight cultures were diluted 1000 fold in fresh LB mediumwith ampicillin (100 �g/ml) and grown until OD600 was 0.5.Isopropyl-�-d-thiogalactopyranoside (IPTG) at a final concentra-tion of 0.1 mM was added to the cultures and the cells were furthergrown until OD600 was1.0. The cells were washed in M9 bufferand subsequently incubated for 15 min or 30 min at 37 ◦C in M9buffer with various concentrations of MNNG (Aldrich) or methylmethanesulfonate (MMS, Sigma). Finally, cells were washed withM9 buffer and spread on LB plates with ampicillin (100 �g/ml) orrifampicin (50 �g/ml) at appropriate dilutions. Surviving colonieswere counted after incubation at 37 ◦C for one or two days.
2.5. Protein expression and purification
Ten liters of E. coli ER2566 carrying plasmid pET-adaB or pET-adaA-alkA were grown at 37 ◦C in LB-kanamycin (50 �g/ml) untilOD600 was 0.5. Next, IPTG was added to the cells at a final con-centration of 0.25 mM and incubation was continued at 18 ◦C overnight. The cells were collected, washed once in cold water, resus-pended in 150 ml extract buffer (300 mM NaCl, 50 mM Na2HPO4, pH7.5) and run through the French Pressure Cell Press (SLM Aminco)at 12000 PSIG twice. The cell-free protein extract obtained aftercentrifugation (15,000 × g for 30 min at 4 ◦C) was loaded on a 3 mlnickel agarose column (Qiagen) equilibrated with extract buffer.The column was washed with 10 ml extract buffer supplementedwith 50 mM imidazole and subsequently eluted with extract buffersupplemented with 300 mM imidazole. The protein samples weresubjected to SDS-PAGE and the protein bands corresponding to theexpected size of AdaB and AdaA-AlkA were verified by mass spec-trometry. Bradford protein assay (Bio-Rad) was used to measureprotein concentration. To ensure that the purified recombinant pro-tein was not contaminated with endogenous methyltransferases
or alkylbase DNA glycosylase, we followed the same purificationprotocol with extracts prepared from ER2566 carrying the emptyvector pET28b. No methyltransferase or alkyl DNA glycosylaseactivity could be detected in the fractions from the Ni-column.
M. Yang et al. / DNA Repair 10 (2011) 595–602 597
Fig. 1. (A) Schematic representation of chromosomal organization of the adaptive response genes in E. coli and the structural homologues in M. tuberculosis. The adaA, adaBand alkA genes are differently composed in E. coli and M. tuberculosis. Whereas adaA is fused with alkA in M. tuberculosis, adaA is fused to adaB in E. coli. The arrows indicatepromoter regions. (B) Predicted methyltransferase functions of M. tuberculosis AdaA-AlkA (upper panel) and AdaB (bottom panel) based on homology to E. coli Ada and AdaB.The AdaA-AlkA protein is predicted to transfer methyl groups from methylphospotriesters of the DNA backbone to a conserved cysteine residue (Cys34) of the AdaA domain,s he adp R = de
2
tTcatuo(ToUNc
2
cDMmu(1bsl(
uggesting that AdaA-AlkA is converted to a positive transcriptional regulator of tredicted to transfer methyl groups from O6-mG in DNA to its conserved Cys 126. d
.6. DNA methyltransferase assay
N-[3H] methyl-N′-nitrosourea (MNU; 1.5 Ci mmol−1) was usedo prepare alkylated calf thymus DNA (6000 d.p.m. �g−1 DNA) [16].he methyltransferase activity was assayed in a reaction bufferontaining 90 mM Tris, 90 mM Borate, 2 mM EDTA (TBE buffer)t 37 ◦C for 60 min. The reaction mixture contained 1.5 �g MNUreated calf thymus DNA and protein as indicated in a total vol-me of 3 ml. Proteins were precipitated by incubating with 100 �lf 10 mg/ml BSA (in TBE buffer) and 1 ml of 4 M perchloric acidHClO4) for 30 min at 70 ◦C, and centrifuged at 4000 rpm for 20 min.he pellet was washed with 4 ml of 1 M HClO4 and resolved in 0.4 mlf 0.1 M HCl. The samples were transferred into tubes containingLTIMA GOLDTM MV scintillation liquid (Packard BioScience B. V.,etherlands), and the radioactivity was measured in a scintillationounter (Liquid Scintillation Analyzer, TRI-CARB 2900TR).
.7. Alkylbase DNA glycosylase assay
The DNA glycosylase activity was assayed in a reaction bufferontaining 50 mM MOPS pH 7.5, 1.0 mM EDTA, 5% glycerol, 1.0 mMTT at 37 ◦C for 30 min. The reaction mixtures contained 0.3 �gNU treated calf thymus DNA (same substrate as for the DNAethyltransferase assay) and protein as indicated in a total vol-
me of 50 �l. The reaction was stopped by adding 73 �l stop buffer0.41 M NaAc, 0.027% carrier-DNA, 0.82 mg/ml BSA) and 350 �l00% ethanol and kept at −80 ◦C for 30 min. DNA was precipitated
y centrifugation at 13,000 rpm for 15 min at 4 ◦C. 350 �l of theupernatant was transferred to UITIMA GOLDTM MV scintillationiquid, and the radioactivity was measured in a scintillation counterLiquid Scintillation Analyzer, TRI-CARB 2900TR).
aptive response to DNA alkylation damage in M. tuberculosis. The AdaB protein isoxyribose.
2.8. Homology modeling of M. tuberculosis AlkA
The model of the C-terminal AlkA domain of M. tuberculosisAdaA-AlkA was built using the Swiss-Model homology-modelingserver [17], and is based on the crystal structure of E. coli AlkAwithout DNA (pdb-code 1mpg). M. tuberculosis AdaA-AlkA has afew inserts compared with E. coli AlkA (Supplementary data Fig.S1B), but they are all located at surface-exposed loops distant fromthe DNA binding groove.
3. Results
3.1. Organization of the ada operon in M. tuberculosis
To defend against fluctuating concentrations of alkylatingagents many bacteria mount an inducible response that enhancestranscription of several genes protecting against alkylating stress.In E. coli, induced alkylation resistance is acquired by the expres-sion of four genes, ada, alkA, alkB and aidB (Fig. 1A). Componentshomologous to the Ada and AlkA proteins in E. coli are fused indifferent combinations in other species. The evolutionary fusion ofAdaA and AlkA appears to be as common as AdaA with AdaB. Byexample, the N-terminal domain of Ada (AdaA) is fused with AlkAin M. tuberculosis (Fig. 1A). Further, the ORF of AdaA-AlkA forms anoperon that also includes a putative AdaB methyltransferase. TheadaA-alkA and adaB (annotated as ogt in the genome sequence) [18]gene sequences overlap by four basepairs. Characteristic sequencesignatures and residues are conserved in both AdaA-AlkA and AdaB.This includes the zinc finger of AdaA, the active site thiol of AdaA(Cys38 in E. coli, Cys34 in M. tuberculosis) and AdaB (Cys321 in E. coli,
Cys126 in M. tuberculosis), the helix-hairpin-helix motifs (HhH)of AdaB and AlkA, the arginine finger of AdaB as well as the cat-alytic aspartic acid (Asp238 in E. coli, Asp441 in M. tuberculosis) ofAlkA (Supplementary Fig. S1A–C). The predicted methyltransferase
598 M. Yang et al. / DNA Repair
*
*
* *0
10
20
30
40
aidBalkBadaBadaA-alkA
Rel
ativ
e m
RN
A le
vel
Control
MNNG
Fig. 2. Adaptive response for inducible repair of DNA alkylation damage in M. tuber-culosis. The cells were grown with or without (control) 3 �M MNNG for 60 min, thetotal RNA were isolated and the transcription levels for adaA-alkA, adaB, alkB andaidB were measured by quantitative real-time PCR. The housekeeping gene sigAwge
aFM
3i
a
Fpwcacro
as used as endogenous control to normalize the relative expression level of targetenes. The data are presented as mean of six measurements from three independentxperiments with standard deviation. *P < 0.01.
ctivities of M. tuberculosis AdaA-AlkA and AdaB are illustrated inig. 1B, in which Ada-AlkA and AdaB transfer methyl groups fromPT and O6-mG in DNA, respectively.
.2. Ada response for inducible repair of DNA alkylation damage
n M. tuberculosis
Regulation of putative alkylation resistance genes, such as adaA-lkA, adaB, alkB and aidB, in M. tuberculosis were examined by real
ig. 3. Functional complementation of the methyltransferase deficient E. coli mutant KT2UC18 and KT233 cells carrying pUC18, pUC18-adaA-alkA or pUC18-adaB were treated were spotted on LB plates and incubated at 37 ◦C. Spot formation was visualized after o
arrying pUC18 or pUC18-adaB were exposed to increasing concentrations of MNNG for 1fter two days of incubation. Each point is the mean three independent experiments witarrying pUC18, pUC18-adaA-alkA or pUC18-adaB were treated with 7 �M MNNG for 1ifampicin. The number of rifampicin resistant mutants were counted after two days of if three independent experiments with standard deviation. *P < 0.01 when compared to t
10 (2011) 595–602
time quantitative PCR on RNA samples collected before and aftertreatment with MNNG. The results showed that adaA-alkA and adaBexpressions were 16 and 27 times upregulated, respectively, inresponse to MNNG, whereas alkB and aidB were moderately upreg-ulated, 2.6 and 1.6 times, respectively (Fig. 2). It thus appears thatan adaptive response for inducible repair of DNA alkylation dam-age at least include adaA-alkA and adaB of the ada operon in M.tuberculosis.
3.3. Functional complementation analysis of the E. coli mutantsBK2118 alkA tag and KT233 ada ogt with the M. tuberculosis adaoperon
To elucidate the characteristics of the ada operon of M. tubercu-losis, plasmid vectors (pUC18) containing adaA-alkA or adaB geneswere transformed into E. coli mutants lacking the alkylbase DNAglycosylases Tag and AlkA (BK2118) or methyltransferases Adaand Ogt(KT233) for functional analysis. Expression of adaB sup-pressed the MNNG sensitivity of the KT233 mutant (Fig. 3A andB), whereas adaA-alkA expression showed no effect on survival(Fig. 3A). This data suggested that AdaB possesses O6-mG trans-ferase activity. Moreover, the mutation frequency, measured asrifampicin resistance, of KT233 expressing AdaB was reduced belowthe wild type level (Fig. 3C), supporting the survival data. In con-trast, expression of AdaA-AlkA showed no change in the mutationfrequency of KT233, indicating that the putative AdaA domain
of AdaA-AlkA is not involved in removal of the mutagenic O6-mG lesion. Surprisingly, survival experiments with BK2118 cellsexpressing AdaA-AlkA or the AlkA domain alone plated on mediacontaining the alkylating agent MMS showed no suppression of
33 (ada ogt) with M. tuberculosis adaA-alkA and adaB. (A) Wildtype E. coli carryingith or without (control) 680 �M MNNG for 30 min at 37 ◦C. Serial dilutions (1–10−4)ne day of incubation. (B) E. coli wild type carrying pUC18 and KT233 mutant cells5 min, spread on LB plates and incubated at 37 ◦C. Surviving colonies were countedh standard deviation. (C) E. coli wild type carrying pUC18 and KT233 mutant cells5 min at 37 ◦C, serially diluted and plated on LB plates with or without 50 �g/ml
ncubation and related to the total number of cells. The data are presented as meanhe KT233 strain carrying pUC18.
M. Yang et al. / DNA Repair 10 (2011) 595–602 599
0.001
0.01
0.1
1
10
100
21.510.50MMS (µM)
Surv
ival
(%)
Wild type/pUC18 BK2118/pUC18
BK2118/pUC-alkA BK2118/pUC-adaA-alkA
Fig. 4. Functional complementation of the DNA glycosylase deficient E. coli mutantBK2118 (tag alkA) with M. tuberculosis adaA-alkA. E. coli wildtype cells carryingpUC18 and BK2118 carrying pUC18-adaA-alkA, pUC-alkA or pUC18 were treatedwci
aAs
3g
fEaw
0
25
50
75
100
AlkDAdaBAdaA-AlkA
Enzyme
Rel
ease
d m
ethy
late
d ba
ses
(fmol
)
Fig. 6. Alkylbase DNA glycosylase activity of recombinant AdaA-AlkA and AdaB.Each protein sample (100 ng AdaA-AlkA or AdaB) was incubated with [3H]-MNUtreated DNA at 37 ◦C for 30 min, and the DNA was precipitated with ethanol. Theradioactivity of the supernatant (released bases) was measured by a liquid scintilla-tion counter. The DNA glycosylase AlkD from Bacillus cereus was included as positivecontrol. The data are presented as mean of three measurements with standard
FwaA
ith increasing concentrations of MMS for 15 min at 37 ◦C. Surviving colonies wereounted after two days of incubation. The data are presented as mean of threendependent experiments with standard deviation.
lkylation sensitivity (Fig. 4), indicating that the AlkA domain ofdaA-AlkA is not involved in repair of cytotoxic N-alkylated basesuch as 3mA and 3mG.
.4. Analysis of methyltransferase and 3-methyladenine DNAlycosylase activity
Recombinant M. tuberculosis AdaA-AlkA and AdaB proteins
used with N-terminal His tags were purified to homogeneity from. coli. To examine DNA methyltransferase activity and removal oflkylated bases, DNA treated with [3H]-labeled MNU was incubatedith protein extracts or purified enzymes. The methyltransferase
A
C
*
*
0
10
20
30
40
pET-adaBpET-adaA-alkAVector
E. coli extract
Tran
sfer
red
met
hyl (
fmol
)
0
5
10
15
20
25
30
1.00.0A
Tran
sfer
red
met
hyl (
fmol
)
ig. 5. DNA methyltransferase activity of recombinant AdaA-AlkA and AdaB. The protein saith perchlorid acid and resolved in HCl. The methyltransferase activity was measured by l
ctivity of cell-free extract (10 �g) prepared from E. coli KT233 carrying pUC18, pUC18-adaAdaA-AlkA and AdaB at increasing concentrations. The data are presented as mean of thr
deviation. *P < 0.01.
activity was monitored as the amount of label present in the proteinfraction. Methyltransferase assays with protein extracts preparedfrom cells overexpressing AdaA-AlkA and AdaB showed signifi-cantly higher transfer of methyl groups as compared to the controlextract (only vector) (Fig. 5A). Next, methyltransferase assays withincreasing amounts of purified enzymes demonstrated that methylresidues were transferred to both AdaA-AlkA and AdaB (Fig. 5B andC). Finally, we tested 3mA DNA glycosylase activity of the puri-fied proteins; however, no excision of alkylated bases could be
detected in AdaA-AlkA or AdaB proteins (Fig. 6). In order to excludethat the N-terminal His-tag interfere with 3mA DNA glycosylaseactivity, we tested activity in extracts from BK2118 carrying plas-
0
5
10
15
20
25
3.02.01.00.0
AdaA-AlkA (pmol)
Tran
sfer
red
met
hyl (
fmol
)
3.02.0daB (pmol)
B
mples were incubated with [3H]-MNU treated DNA at 37 ◦C for 60 min, precipitatediquid scintillation counting of the label in the protein fraction. (A) Methyltransferase-alkA or pUC18-adaB. (B) and (C) Methyltransferase activity of purified recombinant
ee measurements with standard deviation. *P < 0.01.
600 M. Yang et al. / DNA Repair 10 (2011) 595–602
Fig. 7. Modeling the base binding pocket in M. tuberculosis AdaA-AlkA and comparison with E. coli 3-metyl adenine DNA glycosylases I (AlkA) and II (Tag). (A) Crystal structureof active site in E. coli AlkA with abasic DNA (AP-site). Modeling of 3mA in this pocket suggests that the positively charged, alkylated base is stacking face-to-face with Trp272and edge-on with Tyr222. (B) Model of the corresponding pocket in M. tuberculosis AdaA-AlkA. The edge-on stacking residue Tyr222 in E. coli AlkA is replaced by Val425.Several other aromatic residues in AlkA are replaced by smaller amino acid residues in M. tuberculosis AdaA-AlkA: Tyr273 by Thr480, Trp14 by Gly214, Phe18 by His218 andT de bino g as a
meavagds
3
aacoEmstcrstDad3ApFmttAdpclb
4
am
yr239 by Leu442. These sequence changes could result in a more flexible nucleotif E. coli Tag in complex with abasic DNA and free 3mA illustrating aromatic stackin
ids pUC18 or pUC18-Ada-AlkA. The pUC18-Ada-AlkA constructxpresses the native protein, however, no 3mA DNA glycosylasectivity could be detected (data not shown). In view of the sur-ival and mutagenesis data together with the results of the enzymectivity assays, it appears that AdaA-AlkA and AdaB transfer methylroups from MPT’s and O6-mG, respectively. However, direct evi-ence that Ada-AlkA transfer MPT’s is lacking. None of the proteinshowed alkylbase DNA glycosylase activity.
.5. Comparative modeling of AdaA-AlkA
In order to understand the molecular basis for the observedbsence of 3mA glycosylase activity of M. tuberculosis AdaA-AlkA,homology model of the AlkA-like domain was built using the
anonical E. coli AlkA as a template. The C-terminal AlkA domainf M. tuberculosis AdaA-AlkA shares ∼32% sequence identity with. coli AlkA (Supplementary Fig. S1B). A detailed comparison of theodel of M. tuberculosis AdaA-AlkA with E. coli AlkA reveal several
equence discrepancies in residues close to the alkylbase recogni-ion pocket (Fig. 7A and B). N-alkylated bases bear a partial positiveharge, and it has been suggested that a key element in the specificecognition of alkylated bases over native bases involve favourabletacking between the positively charged alkylated base and elec-ron rich, aromatic protein residues. Structures of the E. coli 3mANA glycosylases AlkA and Tag clearly demonstrate this (Fig. 7And C). The residues Trp272 and Tyr222 in AlkA are thought to beirectly involved in such stacking [19], whereas in Tag, the freemA stack with Trp46 (Fig. 7C). Specifically, our model of AdaA-lkA shows that Tyr222 in AlkA is replaced by Val425, whichrobably reduces the affinity toward charged purine substrates.urthermore, compared with the tight stacking of several aro-atic residues in the second and third coordination sphere around
he pocket in AlkA, AdaA-AlkA contains small amino acid chainshat could destabilize or reduce the rigidity of the base pocket.lthough the catalytic residue Asp 441 is conserved in the AlkAomain it is less likely that this residue could obtain a favourableosition for catalysis in lack of a proper recognition pocket. Thisomputer model supports that the AlkA domain of AdaA-AlkA hasost the specificity for alkylated bases but retained the ability toind DNA.
. Discussion
Previous works have suggested that sequence variations in thedaptive response genes of M. tuberculosis lead to transient hyper-utators, which favors host adaptation [12,20]. Here, we have
ding pocket with less affinity for rigid and planar nucleotides. (C) Crystal structuremechanism of recognition of alkylated bases in DNA [28].
characterized the ada operon of M. tuberculosis, which encodes acomposite protein of AdaA and AlkA, and a separate AdaB protein.Exposure of M. tuberculosis to MNNG strongly increased transcrip-tion of adaA-alkA and adaB, demonstrating an inducible responseto methylating agents. Functional complementation assays ofE. coli ada ogt cells showed that only adaB reduced the sponta-neous mutation frequency and rescued the MNNG sensitivity ofthe methyltransferase deficient mutant. Purified AdaA-AlkA andAdaB possessed methyltransferase activity on methylated DNAbut no 3mA DNA glycosylase activity. These data suggested thatthe AdaB protein mediates a methyltransferase activity counter-acting the cytotoxic and mutagenic effect of O6-mG. Further wepropose that AdaA-AlkA transfers methyl groups from innocuousmethylphosphotriesters although direct evidence are lacking. Itthus appears that the AlkA domain of AdaA-AlkA has no role in baseremoval of cytotoxic alkylation lesions such as 3-methylpurinesbut may have evolved a structural function in DNA recognition.Our data suggest that the main role of the adaptive response inM. tuberculosis is to suppress O6-mG alkylation induced mutagen-esis rather than removal of cytotoxic DNA lesions such as 3mA and3mG.
To our knowledge this is the first functional characterizationof the AdaA-AlkA fusion protein family. Surprisingly, AdaA-AlkApossesses no alkylbase DNA glycosylase activity despite the aminoacid conservation with the AlkA family. However, our structuralpredictions indicate that critical residues of the base recognitionpocket are changed to residues repealing the specificity of the AlkAdomain to alkylated bases but still retaining the ability to bindDNA. Consequently, we may speculate whether the AlkA domainof AdaA-alkA has evolved a function required for DNA bindingand scanning the genome for methylphosphotriesters and operatorsequences of the adaptive response genes. However, it remains toprovide experimental evidence that AdaA-AlkA has a potential roleas a regulator of the adaptive response in M. tuberculosis. Notably,the AdaA domain of AdaA-AlkA lacks the C-terminal HhH motif,which is present in AdaA proteins without a fusion partner suchas the AdaA protein of B. subtilis [21]. However, the AlkA domainof AdaA-AlkA contain a HhH motif and several structural studiesof DNA glycosylases and transferases demonstrate that the HhHmotif is required for DNA binding (review by Dalhus et al., 2009)[2]. Therefore, we suggest that the entire AlkA fold of the AdaA-AlkAfusion protein has functionally replaced the C-terminal HhH motif
of the AdaA fold. In addition to the AlkA domain of AdaA-AlkA,the M. tuberculosis genome encodes two putative alkylbase DNAglycosylases, Tag and Aag. The 3mA-DNA glycosylase Tag, whichwas first discovered in E. coli, is widespread in prokaryotes but not
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resent in mammalian cells [22,23]. Aag was first characterized inammalian cells [24], however, Aamodt et al. showed that Bacil-
us subtilis encodes a functional Aag DNA glycosylase [25]. Thus,t appears that M. tuberculosis harbors alternative DNA glycosy-ases, other than AlkA, to remove cytotoxic alkylated bases suchs 3-methylpurines.
Our work demonstrates that MNNG strongly upregulatesxpression of adaptive response genes such as adaA-alkA and adaBn M. tuberculosis. A previous study showed that the alkylating agenttreptozotocin induced O6-mG transferase activity in M. smegma-is, supporting our findings [26]. However, M. bovis showed noegulation of O6-mG transferase activity by streptozotocin [26].nterestingly, the sequence of the M. bovis adaA-alkA gene con-ains a stop codon at the end of the adaA sequence, separatinghe gene into two open reading frames for AdaA and AlkA. We
ay speculate if a functional AdaA-AlkA protein requires the AlkAomain to induce the adaptive response to alkylation damage inycobacteria. To investigate this hypothesis it would be tempting
o introduce a stop codon into the M. tuberculosis ada-alkA genend test if this destroys induction of the methylation inducibleenes.
M. tuberculosis is expected to sustain significant levels ofitrosative stress that form potent DNA alkylating compounds dur-
ng the course of an infection. Durbach et al. constructed a M.uberculosis mutant strain lacking the ada operon, including adaA-lkA and adaB, to address the consequences of alkylation damage20]. The ada mutant showed a 100-fold increase in mutation fre-uency as compared to wild-type cells after treatment with MNNG,upporting that AdaB possesses a methyltransferase activity coun-eracting the mutagenic effect of O6-mG. Moreover, the ada mutantisplayed hypersensitivity to MNNG comparable to the hypersen-itivity of E. coli KT233 (ada ogt) [20], demonstrating the cytotoxicffect of O6-mG. Further, this result is in agreement with our bio-hemical data demonstrating that AdaA-AlkA of the Ada operonn M. tuberculosis possesses no DNA glycosylase activity towardytotoxic alkylating products such as 3mA and 3mG. To examinehether the same M. tuberculosis mutant would be manifested in a
rowth phenotype in vivo, mice were infected with wild type andutant strains and bacillary loads were counted in various organs
20]. However, no differences in bacillary loads were found in anyrgans of the mice infected with the mutant strain as comparedo the wild-type control, indicating that permeation of nitrosativetress to the level of cytotoxic alkylated DNA base lesions is notignificant in adaptive response-defective M. tuberculoses strainsn vivo. In sum, those data and the present work strongly suggesthat the adaptive response to alkylation damage is restricted touppress mutagenesis in M. tuberculosis. Consequently, these find-ngs indicate that pathogenic M. tuberculosis strains with a defectivedaptive response to alkylation may confer a selective advantageontributing to adaptation to the host and environmental changes.n this context, a transient hypermutator phenotype that sustainshe robustness to cytotoxic alkylation DNA lesions can secure cel-ular fitness. Notably, Ebrahimi-Rad et al. reported that nine outf 55 W-Beijing strains had a characteristic mutation on codon7 (Arg to Leu) of the adaB gene [12]. The relative contribution of-Beijing genotype strains to the current worldwide TB epidemic
s increasing, suggesting that the adaptive response genes of theda operon are important targets for further investigations [27].urther, Nouvel et al. sequenced the ada operon in 55 multi-drugesistant (MDR) Central African Republic (CAR) strains and identi-ed three strains with a mutation in adaB (codon15, Thr to Ser) and1 strains with mutations in adaA-alkA (codon 12, Ile to Val; codon
9, Trp to AMBER; codon 337, Thr to Asn) [11]. Moreover, the sametudy report that 34 out of 194 non-MDR CAR strains carry adaA-lkA mutations. In conclusion, our data underscore the importancef the adaptive response in mycobacterial species and suggest that
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the methyltransferases are essential to suppress alkylation inducedmutagenesis in M. tuberculosis.
Funding
This work was supported by grants from The Research Councilof Norway to MB and the European Commission to TT.
Conflict of interest
The authors declare that there are no conflicts of interest.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.dnarep.2011.03.007.
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