Mutation Research

Fundamental and Molecular Mechanisms of Mutagenesis

684:24-34 (2010).

Chloroacetaldehyde-induced mutagenesis in Escherichia coli: the role of AlkB protein in

repair of 3,N4-ethenocytosine and 3,N4-α-hydroxyethanocytosine

Agnieszka M. Maciejewskaa, Karol P. Ruszela, Jadwiga Nieminuszczya, Joanna Lewickaa,

Beata Sokołowskab, Elżbieta Grzesiuka and Jarosław T. Kuśmiereka,*

aInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, 5A

Pawińskiego Str, Poland; bMedical Research Center, Polish Academy of Sciences, 02-106

Warsaw, 5 Pawińskiego Str, Poland

* To whom correspondence should be addressed. Tel.: +48 22 592 3338; Fax: +48 22 592 2190; E-mail: jareq @ ibb.waw.pl .

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Keywords: E. coli AlkB, AlkA and Mug proteins; repair of etheno adducts;

chloroacetaldehyde-induced mutagenesis; LacZ→Lac+ reversion

Abbreviations: CAA, chloroacetaldehyde; ε, etheno, see Fig. 1 for structures and

abbreviations of etheno adducts; MF, mutation frequency; MMS, methyl methanesulphonate.

.

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Abstract

Etheno () adducts are formed in reaction of DNA bases with various environmental

carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde

(CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate -adducts. We

studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of -

adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104

plasmids bearing the lactose operon of CC102 or CC104 origin (C.G. Cupples, J.H. Miller.

Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 5345-5349) which allowed to monitor Lac+

revertants, the latter arose by GCAT or GCTA substitutions, respectively, as a result of

modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and

replicated in E. coli of various genetic backgrounds. To modify the levels of AlkA and AlkB

proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response.

Formation of C proceeds via a relatively stable intermediate, 3,N4--hydroxyethanocytosine

(HEC), which allowed to compare repair of both adducts. The results indicate that all three

genes, alkA, alkB and mug, are engaged in alleviation of CAA-induced mutagenesis. The

frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison

to alkA+, alkB+, and mug+ controls. Considering the levels of CAA-induced Lac+ revertants in

strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that

AlkB protein is engaged in the repair of C and HEC in vivo. Using the modified TTCTT 5-

mers as substrates, we confirmed in vitro that AlkB protein repairs C and HEC although far

less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of

HEC and εC is significantly different from that for 3-methylcytosine. We propose that the

protonated form of adduct interact in active site of AlkB protein.

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1. Introduction

The alkB gene was discovered over twenty years ago in Mutsuo Sekiguchi’s

laboratory while isolating Escherichia coli mutants with increased sensitivity towards

alkylating agents [1]. This gene is a part of the system of adaptive response to alkylating

agents in E. coli (the Ada response), 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 alkylated bases in DNA using different

mechanisms, whereas the function of the aidB gene product has not been conclusively

established (recently reviewed in [2]). Although the phenotypic characteristics of alkB

mutants as well as logical reasoning indicated that 3-methylcytosine (m3C) and 1-

methyladenine (m1A) are the most plausible substrates for AlkB [3], the direct biochemical

evidence emerged only recently. Crucial information in elucidation of the enzymatic function

of AlkB was provided by bioinformatics analysis which established that AlkB is a member of

the superfamily of 2-oxoglutarate- and iron-dependent dioxygenases [4]. Independent studies

in Barbara Sedgwick’s and Erling Seeberg’s laboratories have revealed that purified AlkB

removes methyl groups from m3C and m1A via oxidation to formaldehyde in the presence of

2-oxoglutarate, Fe(II) and O2, thus restoring undamaged bases in DNA [5,6]. Further studies

revealed that analogous alkB genes are present in various organisms including humans, and

that besides methylated ssDNA, also dsDNA and RNA can be substrates for AlkB and its

homologues. However, each protein displays a different repair propensity for particular type

of nucleic acid. In addition to m3C and m1A, the structurally similar lesions, 1-methylguanine

and 3-methylthymine are also repaired, albeit less efficiently, by human and bacterial AlkB

proteins. Moreover, it was found that AlkB protein removes from DNA not only methyl

groups but also higher alkyl homologues (for latest reviews see: [2,7-9]).

The etheno (ε) base adducts (see Fig. 1 for structures) constitute another, structurally

distinct from linear alkyl base adducts, group of AlkB substrates. They belong to exocyclic

DNA base adducts formed by various bifunctional agents of exogenous and endogenous

origin. They are generated by many industrial carcinogens: e.g., vinyl chloride metabolites,

chloroethylene epoxide and chloroacetaldehyde (CAA). The oxidative stress driven lipid

peroxidation is a powerful source of endogenous reactive agents, which react with DNA to

form etheno and other types of adducts, and accumulating evidence suggests that these types

of lesions play a role in the etiology of some human cancers [10].

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It has been found that the toxic and mutagenic effect caused by the presence of εA and

εC in site-specifically modified M13 viral DNA in E. coli alkB mutant was alleviated in alkB+

cells. Also, in vitro experiments have shown that the etheno bridge of εA, situated in an

oligodeoxynucleotide, is removed by AlkB protein via epoxidation of the double bond and is

finally released as glyoxal [11]. Similarly, the in vitro repair of εA by bacterial AlkB and its

human homologue hABH3 has been demonstrated by another group [12]. 1,N6-

ethanoadenine, an analogue of εA, containing a saturated two-carbon bridge formed through

the reaction of adenine in DNA with the antitumor agent 1,3-bis(2-chloroethyl-1-nitrosourea

(BCNU), upon action of AlkB protein is transformed into a non-toxic and weakly mutagenic

N6-2-oxoethyl derivative [13]. AlkB also influences CAA-induced mutation spectra and

toxicity in the pSP189 sup F shuttle vector, which implies AlkB contribution to repair of ε-

adducts and other exocyclic adducts generated by CAA [14]. Recently, it has been shown that

hABH2, but not hABH3, is active in the direct reversal of εA, and that mABH2 is the

principal dioxygenase for εA repair in mice [15].

CAA is highly toxic and weakly mutagenic agent. This CAA feature makes studying

CAA mutagenesis in bacteria very difficult. Here, using a new method that allows to examine

the mutagenic potency of CAA but eliminates its cytotoxic action on the cell, we studied the

role of AlkB protein in CAA-induced mutagenesis and repair of ethenoadducts in DNA. The

test system used involved pIF102 and pIF104 plasmids [16] containing the lactose operon of

the CC102 or CC104 strain [17]. The plasmid DNA was modified with CAA in vitro, but

DNA repair processes were studied in vivo in live bacterial cells. Transformation of CAA-

pretreated plasmid DNA into E. coli cells allowed to study GCAT transitions (pIF102) and

GCTA transversions (pIF104) that can arise by the modification of both components of the

G·C pair, guanine and cytosine. The GCAT transition dominates among base substitutions

caused by CAA in E. coli cells [18]. Similarly, GCAT transitions followed by GCTA

transversions are the most frequent base substitutions in CAA-treated shuttle vector

propagated in the cultured human fibroblast cells [19]. It is worth to mention that GCAT

transitions are the most frequent mutations in MMS-treated alkB strains [20]. Since the

reaction of cytosine moiety with CAA proceeds via a relatively stable intermediate, 3,N4--

hydroxyethanocytosine (HEC, Fig. 1), we attempted to compare the mutagenic potential and

repair of both cytosine adducts, HEC and a stable product of its dehydration, εC.

The use of wild type E. coli and mutants deficient in AlkB, AlkA and the mismatch

uracil-DNA glycosylase Mug, all engaged in repair of ε-adducts, allowed to estimate the role

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of each enzyme in DNA repair. We also present results indicating that purified AlkB protein

repairs CAA-modified cytosines in oligodeoxynucleotides.

2. Materials and Methods

2.1. Bacterial strains

The E. coli K12 strains used in this study are listed in Table 1. The strains made for

mutagenesis tests were constructed by P1-mediated transduction according to [21]. The AM1

strain was a recA+ DH5α derivative constructed by transduction of srl::Tn10, recA+ from

EC1651 and selected for tetracycline resistance and loss of UV sensitivity. The obtained

strain (DH5α recA+, srl::Tn10) was a recipient of srl+ gene from the AM100 strain (with

selection for sorbitol as the only source of carbon). AM4 was also constructed in two steps.

First, Tn10 zee3129, his was transferred from EC2445 to AM1 (with selection for tetracycline

resistance followed by screening for histidine requirement). Then, to the resultant AM1 Tn10

zee3129, his strain, the alkA1 point mutation was co-transduced with the his+ gene from the

MS23 strain. Transductants were selected on glucose minimal plates, and subsequently

screened for loss of tetracycline resistance and enhanced MMS sensitivity. Details of

construction of all AM1 derivative strains are given in Table 1. The alkB117 and mug-1 were

selected on LB plates [21] containing 50 μg/mL carbenicillin or 50 μg/mL kanamycin,

respectively. At every step the transductants were checked for enhanced MMS and CAA

sensitivity.

2.2. Media

Bacteria were cultured in rich LB [21] and SOC [25] media. Minimal medium (MM)

was C-salts [26] supplemented with 0.4% lactose, or 0.5% glucose, or 0.5 % sorbitol and

thiamine (10 g/ml) if necessary. LB and MM were solidified by the addition of 1.5% Difco

agar.

2.3. CAA modification of pIF102 and pIF104 plasmids

Plasmids were isolated according to standard protocol [25] and then treated with CAA

at 37°C for 30 min. The incubation mixture in the volume of 0.4 mL contained: 100 μg of

plasmid DNA, 300 mM sodium cacodylate buffer pH 7.5, and 2.9 – 29 μL CAA (Merck, 45%

aq. solution, 6.88 M, final concentration 50 – 500 mM, respectively). After modification DNA

was precipitated twice with 2.5 volumes of ice-cold ethanol in the presence of 0.3 M sodium

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acetate buffer pH 5.2, washed with cold ethanol, redissolved in appropriate volume of TE

buffer and then frozen at -20ºC. The CAA-modified plasmids prepared this way contained the

majority of cytosine-CAA adduct in the form of HEC (plasmid pIF102-HEC and pIF104-

HEC). To convert the HEC intermediate to εC, the plasmid solutions were brought to pH 6.5

by addition of 0.8 M sodium cacodylate buffer pH 6.5 to a final concentration of 20 mM and

incubated at 37°C for 72 h (plasmid pIF102-εC and pIF104-εC) [27]. Mock treated plasmids

were prepared in the same way except that CAA was absent from the incubation mixture. The

mutagenicity assay experiments comparing HEC- and εC-containing plasmids were always

performed in parallel using plasmids from the same batch of CAA modification.

2.4. Mutagenicity assay: induction of adaptive response, preparation of

electrocompetent cells, transformation by electroporation

Bacterial strains were cultured in two repeats (37ºC, anaerobic conditions, 250 rpm).

When the cultures reached OD600 = 0.4, to one of them MMS (Sigma) was added to a final

concentration of 7.5 mM in order to induce Ada response. Subsequently both cultures were

incubated for 15 min and then were used for preparation of electrocompetent cells according

to standard procedure [25]. The competent cells (50 μL) and 0.5 μg of modified plasmid DNA

were electroporated in a 2-mm ice-cold cuvette at 2.5 kV/cm using the default settings of an

Eppendorf Electroporator 2510 (Eppendorf, Madison, WI). The recovery medium SOC was

added to a 1 mL final volume and then the transformation mixture was incubated at 37°C for

2 h under anaerobic conditions. Appropriate dilutions of the mixture were spread on LB plates

containing chloramphenicol (30 μg/mL) to count the total number of transformed cells and on

lactose MM plates for selection of mutants. Plates were incubated at 37°C and counted after

24 and 72 h for transformation efficiency and for mutagenesis, respectively. Transformation

efficiency was defined as the number of colony forming units (cfu) produced by 1 μg of

plasmid DNA in a transformation reaction. Mutation frequency (MF) was calculated as the

number of mutants per 104 transformed cells. The results of mutagenicity assays for CAA-

treated plasmids pIF102 and pIF104 are visualized in Figs 2 and 3, respectively, and are given

numerically in Supplementary Tables 1 - 4 (see Supplementary Materials).

2.5. Statistics

Data were presented as mean and standard deviation (SD). Analysis of variance

(ANOVA) on ranks with Kruskal-Wallis test was applied. Differences between the groups

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were compared using the Mann-Whitney U test. The statistical evaluation was performed

using a commercial statistical package (Statistica 5.1, StatSoft, Poland).

2.6. CAA and MMS modification of TTCTT oligodeoxynucleotide (T/C 5-mer)

Modification with CAA. To a solution of 220 nmol of the T/C 5-mer (Metabion,

Martinsried, Germany) in 176 μL of 1.3 M triethylammonium bicarbonate (pH ~7.5) 16.4 μL

of 6.88 M CAA (45% aq. solution, Merck) was added, and the mixture was incubated at 37°C

for 3 h. The reaction mixture containing unreacted T/C, T/HEC and T/C 5-mers was purified

from CAA and reaction buffer, and fractionated using preparative HPLC (see Section 2.10).

The early eluting fractions containing T/C and T/HEC 5-mers were lyophilized, redissolved in

TE buffer and used as HEC-substrate (85.7 % of T/HEC 5-mer) for the AlkB assay. Fractions

containing a mixture of modified and unmodified T/C 5-mers, obtained in the other CAA

reaction, were combined, lyophilized, redissolved and incubated (37°C, pH 6.5, 72 h) for

conversion of HEC to εC (see Section 2.3). This material was used as εC-substrate (94.5 % of

T/C 5-mer) for AlkB assay. The representative analytical HPLC profiles of HEC- and εC-

substrates are shown in Fig. 4. Notice that the work-up procedure (lyophilization, storage at -

20°C, etc.) of HEC-substrate does not produce an appreciable amount of T/εC 5-mer. The

identity of HEC- and εC-substrate was ascertained by HPLC analysis after enzymatic

hydrolysis to deoxynucleosides according to [27] (not shown).

Modification with MMS. To a solution of 2.5 nmol of the T/C 5-mer in 52 μL of 0.77

M triethylammonium acetate pH 6.5, 2 μL of MMS (Sigma) was added, and the mixture was

incubated at 37°C for 18 h. The mixture (72% of T/m3C 5-mer by HPLC analysis, not shown)

was used as m3C-substrate for AlkB assay without any further purification.

2.7. AlkB dioxygenase purification

His-tagged E. coli AlkB protein was overexpressed in E. coli BL21(DE3) (Ivitrogen)

carrying pET28balkB plasmid (a generous gift from the late prof. E. Seeberg laboratory

(University of Oslo, Norway)) in 2 L of bacterial culture in LB medium. Expression was

induced with 0.5 mM IPTG for 4 h. Bacterial pellets were sonicated in ice-cold binding buffer

(20 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 10 mM imidazole, 10 mM -

mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride) and centrifuged. The extracts were

loaded onto a 1 ml Ni-NTA column (HiTrap Chelating HP, Amersham – Pharmacia Biotech),

and eluted by imidazole gradient with final imidazole concentration of 500 mM. Fractions

containing purified protein were dialyzed into 20 mM sodium acetate buffer pH 5.0,

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containing 300 mM NaCl and 1 mM DTT; glycerol was subsequently added to final

concentration of 50% and the protein was stored in -70°C.

2.9. AlkB assay

Our observations and also those of others [6] indicate that the presence or

absence of ascorbate in the reaction mixture does not influence the repair activity of AlkB.

Since ascorbate produces a massive peak in HPLC profile, we omitted this reagent in our

assays.

HEC- and εC-substrate. The reaction mixtures in a volume of 20 μL contained: 35

mM sodium acetate buffer pH 3.8-5.0 (the most efficient repair determined after 30 min of

reaction was at pH 4.6), 1 mM dithiotreithol, 0.2 mM Fe(NH4)2(SO4)2, 0.35 mM α-ketoglutarate, 500 pmoles of CAA-modified T/C 5-mer substrate and 50 pmoles of purified

AlkB protein. Reactions were carried out at 37°C for various periods of time and then

analyzed by HPLC. The representative separations are shown in Fig. 4. The pH-dependence

and the time course of repair are shown in Fig. 5A and 5B, respectively.

m3C-substrate. The reaction mixtures in 20 μL volume contained: 50 mM Tris/HCl

buffer pH 7.2-8.8 (the most efficient repair determined after 30 min of reaction was at pH

8.0), 1 mM dithiotreitol, 0.05 mM Fe(NH4)2(SO4)2, 0.5 mM α-ketoglutarate, 500 pmoles of

m3C-substrate and 6.25 pmole of purified AlkB protein. Reactions were carried out and

analyzed by HPLC as in the case of HEC- and εC-substrates. The pH-dependence and the

time course of repair are shown in Fig. 5A and 5B, respectively.

2.10. HPLC chromatography

HPLC was performed using a Waters dual pump system with a tunable UV/visible

light absorbance detector managed by Millenium 2010SS (version 2.15) controller. Analytical

and preparative separations were performed, respectively, on a Waters Nova-Pak® C18, 60 Ǻ,

4 μm, 4.6 × 250 mm, cartridge column, at a flow rate of 1 mL/min, and on HR C18, 60 Ǻ, 6

μm, 3.9 × 300 mm, at a flow rate of 1.75 mL/min. In both cases a linear gradient of 20 mM

triethylammonium acetate pH 6.5 × 30% aq. MeOH over 30 min was applied and detection

was carried out at 270 nm.

3. Results

3.1. DNA reaction with CAA – the estimation of level of base modification.

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The plasmid DNA was CAA-modified essentially under the same conditions as

described by Kim et al.[14]. Therefore, we expect similar level of modification of plasmid

DNA as in the cited work. The number of adducts per 100 nucleotides for 0.6 M CAA dosage

and 1h reaction determined by LC-MS/MS method was as follows: 1,N2G – 0.5, C – 1.5

and A – 2.5. The authors did not measure the level of N2,3G. Our former studies indicate

however, that in dsDNA N2,3G is formed with efficiency comparable to that of A [28]. Also

in that work we have found that C is formed with highest efficiency among the etheno-

adducted bases, whereas Kim et al. [14] reported lower efficiency of C than A. The

discrepancy between these results comes probably from the fact that LC-MS/MS method did

not include the hydrated form of C, namely HEC [14]. In our study CAA-modified DNA was

acid-depurinated, what caused simultaneous conversion of HEC to C, then enzymaticaly

hydrolyzed. The resulted pyrimidine deoxynucleosides were analyzed using HPLC with UV

detection, therefore the determined level of C represented the sum of initially formed HEC

and product of its dehydration, C [28].

Under physiological conditions the half-life of HEC is ~1 day [29]. HEC is enough

stable to detect it in enzymatic hydrolysates of CAA-treated oligonucleotides [27]. As it has

been mentioned in Section 2.6 the work-up procedure (lyophilization, storage at - 20°C, etc.)

of TT(HEC)TT does not produce an appreciable amount of TT(εC)TT 5-mer. The complete

conversion of HEC to εC can be achieved by 72 h incubation at pH 6.5 in 37º C [27].

Therefore, we used pIF102 and pIF104 plasmids freshly reacted with CAA (or stored frozen

at -20ºC) which presumably contained the majority of cytosine-CAA adduct in the form of

HEC (pIF102-HEC and pIF104-HEC) and the same plasmids with HEC dehydrated to C by

prolonged incubation at pH 6.5 (pIF102-C and pIF104-C).

3.2. Transformation efficiency and calculation of mutation frequency

Transformation efficiency of bacterial cells with non-treated (control) pIF102 and

pIF104 plasmids varies within the range of 1105 – 1106 cfu/μg DNA, depending on the

batch of competent cells. These values are 1-2 orders of magnitude lower than the reported

6107 cfu/μg DNA for transformation with pSP18 [14]. This difference can be attributed to

the fact that pIF plasmids (15 Kb) are three times larger than pSP189 (5 Kb), and hence

their effective insertion into bacterial cells is more difficult. The treatment of plasmids with

CAA causes a dose dependent dramatic decrease in transformation efficiency to 5103 and

5102 for single and double mutant strains, respectively, for the highest CAA concentrations

used.

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The efficiency of transformation was measured by chloramphenicol resistance (cmR).

Bacterial cells carrying plasmids with damaged cmR gene cat (chloramphenicol acetyl

transferase) do not grow on LB plates supplemented with chloramphenicol. We suppose,

however, that the real “survival” of plasmids in mutagenesis tests can be higher than that

measured by cmR since a pool of plasmids with damaged cat gene and mutated in β-

galactosidase active center can still grow on lactose as the only source of carbon. The

formally calculated mutation frequencies for highly CAA-damaged plasmids were often

extremely high. This indicates that MF values calculated on the basis of cmR cannot be treated

unconditionally. Therefore, we limited the analyses of our results to those obtained for CAA

concentrations not higher than 200 and 500 μM for pIF102 and pIF104, respectively. Because

of the observed non-commensurability between cmR and the number of mutants, we present

our results showing dependence of mutation frequency on CAA concentration in the form of

bars, and not in the form of linear graphs (Figs. 2 and 3).

3.3. Background mutations level

The results of our studies are visualized in Figs. 2 and 3 and given in numbers in

Supplementary Tables 1-4 (see Supplementary Materials). The MF values for controls (0 mM

CAA) vary depending on the type of plasmid and its incubation for 3 days at 37º C or lack

thereof (dehydration of HEC to εC in the case of CAA-treated plasmids), the bacterial strain,

and employing adaptation procedure to bacterial cells or omitting it, from 0.2 ± 0.1 10-4

(pIF104-HEC, wt strain, adapted) to 82.8 ± 25.4 10-4 (pIF102-HEC, alkAmug strain,

adapted). The higher background mutations level in pIF102 plasmid versus pIF104 is clearly

noticeable. Also the background mutations level is in general in growing order when

mutations in plasmids are scored in wild type, single and then double mutant strains. This

trend is consistent with expectation since in bacterial strains deficient in DNA repair enzymes

the repair of various endogenous DNA lesions should be impaired with resultant higher level

of background mutations.

Since prolonged incubation of plasmid DNA at 37º C, which in the case of CAA-

treated DNA was applied for dehydration of HEC, causes also deaminations and

depurinations of unmodified bases, it should increase the level of background mutations.

Unexpectedly, in majority of tested E. coli strains we observed a decrease in GCAT

background mutations in incubated pIF102 plasmid during its replication. This decrease is

even observed in the majority of Mug-deficient strains lacking a glycosylase responsible for

excision of uracil, a lesion formed during hydrolytic deamination of cytosine and leading to

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C→T substitutions. On the other hand, in the case of the pIF104 plasmid, the background

mutations level increases upon incubation in practically all strains tested. The induction of

adaptive response procedure, which involves preincubation of bacterial cells with MMS, does

not have a clear impact on the level of background mutations in both plasmids tested.

Irrespectively of the fact that the observed background mutations level changes to a

great extent depending on various experimental circumstances and that a reasonable

explanation of this variability is sometimes missing, in each series of experiments the level of

CAA-induced mutations was always significantly higher than that of background mutations,

even at the lowest concentration of CAA tested.

3.4. The frequencies of CAA-induced mutations

The MF of CAA-treated pIF102 and pIF104 plasmids scored in E. coli cells of various

genetic backgrounds are presented in Figs. 2 and 3 (see also Supplementary Tables 1-4). In all

strains tested, with and without induction of Ada response, the increase of CAA concentration

used for plasmid modification results in increase of MF. In general, the MF measured without

induction of Ada response in single mutants are up to 5-fold higher than in wild type bacteria.

In bacteria deficient in two repair proteins the further increase of MF is observed, although

the differences between wt and single mutants are more pronounced than between single and

double mutants. The effects of induction of Ada response on MF in both plasmids replicated

in bacteria of various genetic background are analyzed in Discussion (Section 4.2)

3.5. Repair of εC and HEC by AlkB protein

The repair of C and HEC in comparison to the repair of m3C by AlkB protein was

studied using CAA- and MMS-modified TTCTT 5-mers, respectively, as enzyme substrates.

In preparation of the substrates we exploited the relative inertness of thymine residues in

oligodeoxynucleotides towards various chemical modifiers [27]. This approach was applied in

other laboratories much earlier for preparation of TT(C)TT [30] and more recently for

preparation of T(m1A)T [31]. The use of short oligonucleotides for study the AlkB activity

has an advantage over the other methods since the modified short oligomer can be easily

separated from the unmodified (repaired) one, which allows direct monitoring of repair by

HPLC with UV detection (see Fig.4).

We found that the optimal conditions for repair of C and HEC were significantly

different from that for m3C, especially the pH optima which were 4.6 for cytosine-CAA

adducts and 8.0 for m3C (for other details see Section 2.9). Representative HPLC profiles

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showing repair of TT(HEC)TT and TT(C)TT are presented in Fig. 4. The pH-dependence

and the time course of adducts repair are given in Fig. 5A and 5B, respectively.

4.Discussion

4.1. Molecular background of CAA-induced mutations

The relative efficiency of formation of CAA-induced -adducts in dsDNA in vitro was

found to be: C > A ≥ N2,3G >>> 1,N2G, indicating that 1,N2G is a minor product [28].

The reaction of cytosine with CAA proceeds via a relatively stable intermediate, 3,N4--

hydroxyethanocytosine (HEC), which undergoes spontaneous dehydration forming stable C.

Analogous 1,N6--hydroxyethano intermediate, although much less stable, is also formed in

the reaction of adenine [29,32]. A rational presumption can be made that the reaction of

guanine with CAA proceeds via analogous N2--hydroxyethano intermediates, subsequently

transformed into N2,3G and 1,N2G. However, these intermediates were not reported,

probably because of their instability.

The major guanine adduct, N2,3G, causes exclusively GA transitions [33-35]. The

minor adduct, 1,N2G, creates a strong block to DNA polymerases and shows ambiguous

miscoding behavior [36,37], therefore its contribution to the level of GA or GT mutations

seems to be negligible. Most mutations caused by C and HEC are CT transitions followed

by CA transversions [30,38,39]. The GCAT transitions measured in pIF102 plasmid arise

primarily as the effect of formation of N2,3G but also C and its precursor HEC, however, it

cannot be distinguished whether they are the result of modification of guanine or cytosine. In

contrast, the level of GCTA transversions, scored with the use of the pIF104 plasmid, can

be considered as a result of cytosine modification only.

The main aim of this work was to establish the role of AlkB protein in CAA-induced

mutagenesis and in repair of -adducts in DNA. It is known that -adducts are repaired via

base excision repair. N2,3G is excised from DNA by AlkA glycosylase [18,40] whereas C

and HEC by Mug glycosylase [22,27,41]. 1,N2G can be also excised by Mug, albeit much

less efficiently than C [42]. Therefore, to assess the role of AlkB in repair of the CAA-

induced damage, we used E. coli alkB, alkA and mug mutants. Since AlkA and AlkB are

inducible within Ada response proteins, we compared mutagenesis in non-adopted and

adopted with a low dose of MMS E. coli cells.

The alleviation of CAA-induced mutagenesis in E. coli cells by induction of the Ada

response was established several years ago [18,43] . We postulated that C and HEC are

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excised by AlkA protein in adapted E. coli cells [43]. Soon afterwards, it became known that

these cytosine-CAA adducts are excised from DNA by a different type non-inducible, U/T·G

mismatch specific E. coli Mug or human TDG, glycosylase [27,41,44]. After establishing the

role of AlkB protein in the repair of damaged DNA, we came to the conclusion that inducible

AlkB protein can be a plausible candidate for repair of cytosine-CAA adducts.

In uninduced state the level of AlkB and Ada proteins in E. coli is low and equal 2 to 4

molecules/cell, whereas of AlkA protein is much higher (50 molecules/cell). The induction

of the Ada response results in 100–1000-fold increase in the level of Ada proteins [45,46].

MMS used to induce the Ada response is also a potent inducer of SOS system [47,48]. It is of

interest that induction of SOS is not needed to manifest mutagenic propensity of A and C,

however, mutation fixation at -adduct site in E. coli strains, also in those deprived of SOS

functions, is significantly enhanced by pretreatment of bacterial cultures with UV, alkylating

agents or hydrogen peroxide. This phenomenon, called UVM (UV modulation) response, was

observed in numerous experiments although its molecular mechanism remains unknown [49].

Bearing all of this in mind, we could expect that treatment of E. coli cells with low dose of

MMS induces the antimutagenic Ada response and promutagenic SOS and UVM responses,

especially in alkB mutants [50]. It is very likely that the possible competition of these

contradictory acting factors influences the clarity of results of some of our experiments (see

below).

4.2. Mutagenicity and repair of cytosine- and guanine-CAA adducts in E. coli cells

The results obtained with pIF104-εC (Fig. 2A) clearly show that the products of all the

three studied genes, alkA, alkB and mug, are engaged in repair of εC and guanine-CAA

adducts. The MF in single mutants without induction of the Ada response is 1.5- to 5-fold

higher than in the wt strain, indicating that AlkA and AlkB proteins at the constitutive level

exert observable repair activity. The protective effect of alkB and alkA gene products on

survival of uninduced CAA-treated E. coli cells was also observed [11]. Interestingly, CAA

itself does not induce the adaptive response [18]. Similarly, a reduced mutagenesis and

increased survival of CAA-treated pSP189 was observed in AlkB-proficient E. coli cells in

comparison to AlkB-deficient ones, without prior adaptation [14].

In adapted wt strain and single mutants, a statistically significant decrease in MF was

observed in comparison to non-adapted bacteria. In the double mutants the MF values were

elevated in comparison to single mutants, in both, adapted and non-adapted E. coli cultures.

The alkAmug strain, where the alkB gene is active and can be “turned on” by MMS, showed a

14

significant decrease in MF upon adaptation. This observation emphasizes the role of AlkB in

repair of CAA-induced lesions. Although in alkBmug mutant the adaptation decreases the

MF, the difference between adapted and non-adapted cells is much smaller than in the

alkAmug strain and rather statistically insignificant. This can be explained by the fact that εC

repaired by AlkB, but not by AlkA, is more abundant than N2,3G, and efficient repair of εC-

borne mutations overshadows the effect of N2,3G repair by AlkA. On the other hand, it

cannot be excluded that AlkB repairs both, εC and N2,3G, lesions and hence its

antimutagenic activity is more pronounced in the alkAmug than the N2,3G-specific AlkA

activity in the alkBmug mutant.

The results obtained with pIF102-HEC (Fig. 2B) concern the mutagenicity and repair

of a mixture of cytosine-CAA adducts, consisting of HEC, as the major adduct, and of some

εC. The other contribution to the GCAT substitution, similarly as in pIF102-εC, comes

mostly from N2,3G. However, possible participation of presumable N2-α-hydroxyethanoG

precursor(s) cannot be fully excluded. In wt strain and single mutants, the significant

adaptation effect in AlkB-proficient (wt, alkA and mug) and lack of this effect in AlkB-

deficient strains strongly argue in favor of AlkB being the principal repair enzyme of HEC.

The results obtained in alkAmug mutant, where the adaptation of bacterial cells does not

increase the expected antimutagenic activity of AlkB, can be interpreted as an effect of

simultaneous induction by MMS of promutagenic SOS and UVM responses. The observed

significant effect of adaptation in AlkA-proficient alkBmug mutant leads to the conclusion

that if AlkB and Mug proteins are not active, the effect of excision of G-CAA adducts by

AlkA is clearly visible.

In experiments with pIF104 plasmid monitoring GCTA transversions we presume

that CA substitution arising from cytosine modification is the primary mutation scored (see

Section 4.1). The results obtained with pIF104-εC (Fig. 3A) show that in each AlkB-

proficient strain tested (wt, alkA, mug and alkAmug) the effect of induction of AlkB protein on

MF is statistically significant, whereas in AlkB-deficient strains (alkB, alkBmug and

alkAalkB) such an effect is not observed. This again emphasizes the role of AlkB protein in

repair of εC. The observed very large increase in MF values upon Ada response induction in

the case of alkAalkB and alkBmug strains could again be attributed to the activity of co-

inducted promutagenic SOS and UVM responses.

The MF values observed in the alkA mutant were lower than in the wt strain

independently on the induction of Ada response (Fig. 3A). This unexpected reversed

dependence is highly reproducible and statistically significant. A possible explanation of this

15

can be that AlkA binds C and hinders access of Mug and AlkB which could repair this

adduct. Such binding and then prevention of repair of C has been shown in vitro for ANPG

protein, a functional human analogue of AlkA. Although AlkA protein was found to be a

rather weak C binder in vitro [51], the situation could be different in bacterial cells. The lack

of such reversed dependence in experiments with pIF102-C (Fig. 2A) can be explained by

efficient AlkA-mediated repair of N2,3G, which cannot be observed in pIF104-εC

monitoring C→A.

The findings on possible repair of HEC are presented in Fig. 3B. The effect of

adaptation is seen in AlkB-proficient wt, alkA and mug strains, although in the case of the

alkA strain it does not reach statistical significance. The very clear and statistically significant

effect of adaptation visible in AlkA- and AlkB-proficient mug mutant strongly argue in favor

of AlkB being responsible for repair of HEC because AlkA does not repair this lesion [27].

Unfortunately, in the case of the alkAmug mutant, like in experiments with the same strain

using pIF102-HEC, we did not observe the effect of adaptation.

4.3. Repair of εC and HEC by AlkB protein in vitro

We studied the repair of C and HEC by AlkB protein using CAA-modified TTCTT

5-mers as enzyme substrates. The question raised whether a short oligomer can mimic a long

DNA molecule in its interaction with the AlkB molecule. Studies of this issue has revealed

that although methylated poly(dA) (Km = 1.4 μM and kcat = 11.7 min-1) is a better substrate for

AlkB than the T(m1A)T trimer (Km = 2.8 μM and kcat = 7.4 min-1), the difference is not

dramatic [31]. On the other hand the comparison of repair of m3C and m1A centrally situated

in 3-mers and 5-mers showed that the Km values are 20- to 80-fold higher for 3-mers than for

5-mers indicating that AlkB binds the longer substrates much better [52]. This is consistent

with the observed interactions in the crystal structures of AlkB cross-linked to double-

stranded DNA 13-mers, which show protein contacts spread over the pentanucleotide

sequence containing the alkylated target base [53].

Our preliminary experiments have revealed only marginal repair of C and HEC in

comparison to the m3C. Therefore, we initiated experiments to establish the optimal

conditions for repair of these adducts. We found that the pH optimum for of C and HEC

repair was 4.6, whereas for m3C, 8.0 (Fig. 5A). The efficient repair of A occurs within pH

range 4 – 6 and then substantially decreases at higher pH values [12]. In addition, we

observed that C and HEC were repaired faster at higher Fe(II) and lower α-ketoglutarate

concentrations than m3C (see Section 2.9). The observed dramatic difference in pH optima for

16

repair of C, A and HEC versus m3C suggests that the protonated form of substrate is needed

for proper binding to AlkB active center. The pKa value for m3C is 8.7 [54], whereas for C,

A and HEC these values are 3.7, 3.9 [55] and 6.9 [ 32], respectively (the pKa values for

modified ribonucleosides were compared). Thus, the apparent pH optima for repair

correspond to the pKa values of substrates.

On the basis of the resolved structures of AlkB protein co-crystallized with T(m1A)T

and T(m3C)T substrates [52,56] the authors postulate, among other interactions in enzyme

active center, the formation of an H-bond by the exocyclic NH2 group of m1A and of m3C,

with carboxylic group of Asp135. Such interactions are not possible in the case of m1G and

m3T which explains higher efficiency of AlkB in demethylation of the first two. Since the

exocyclic nitrogen in neutral C or A does not possess a hydrogen atom to form an H-bond,

we postulate that the interaction between Asp135 and the adduced base in enzyme active site

is rather based on electrostatic interaction between positively charged protonated adduct

residue and negatively charged carboxylate group of Asp135.

The time course of repair of HEC, C and m3C are presented in Fig. 5B. Repair of m3C

is completed within 10 min whereas repair of HEC and C, with AlkB concentration being 8-

fold higher, does not reach completion even after 2 h. This indicates that CAA-cytosine

adducts are by far less susceptible to repair than m3C even under optimal conditions for repair

of each type of substrate by the AlkB enzyme. This observation raises the question about

repair of these DNA lesions in vivo where the conditions of enzymatic reaction, such as pH or

Fe(II) concentration, are not optimized in order to fulfill the substrate requirements. Our

results and those obtained in other laboratories [11,14] indicate that in E. coli cells, even

without induction of Ada response, the AlkB repair of -adducts is quite efficient. We

suppose that in vivo other, yet unknown, factors are involved in AlkB-mediated direct reversal

of -adducts.

5. Conclusions and perspectives

Using different experimental approaches, we confirmed other laboratories findings

that the alkB gene, along with other two studied genes, alkA and mug, is engaged in avoidance

of CAA-induced mutagenesis in E. coli. In contrast to other authors studying repair of etheno-

adducts in non-induced bacteria we have shown that induction of adaptive response enhances

the capability of bacterial cells to overcome the mutagenic effect of CAA. The results of the

study of the GCTA mutation which primarily arose from cytosine modification indicate that

both cytosine-CAA adducts, the initially formed 3,N4--hydroxyethanocytosine and the

17

product of its dehydration, 3,N4-ethenocytosine, are repaired by AlkB dioxygenase in

bacterial cells. The direct reversal of these lesions by AlkB protein was also confirmed in our

in vitro experiments. The exocyclic hydroxyalkano adduct of cytosine is a new type of

substrate, structurally distinct from the known AlkB substrates. Therefore, we suppose that

the substrate specificity of AlkB-type proteins may be broader and that these proteins can be

engaged in reversal of various kinds of DNA lesions, also those caused by lipid peroxidation

products in mammalian cells. This emphasizes the possible role of human AlkB homologs in

cancer etiology and cancer preventing.

Acknowledgements

We would like to thank Drs. Iwona Fijałkowska and Piotr Jonczyk from our Institute

for kindly providing the pIF102 and pIF104 plasmids for mutagenesis tests and also the late

Dr. Erling Seeberg for a kind gift of the plasmid for AlkB protein expression. We also are

indebted to Ms. Anna Karcz for skillful participation in performing of numerous mutagenesis

experiments. This work was supported by grant N301 065 31/1979 of the Ministry of Science

and Higher Education and partly by the Polish-Norwegian grant PNRF-143-AI-1/07.

18

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22

Figure legends

Fig. 1. Structures of chloroacetaldehyde (CAA) adducts to DNA bases.

Fig. 2. Frequencies of GC→AT mutations induced by CAA in the lacZ gene of the pIF102

plasmid replicated in wild type and various AlkA-, AlkB- and Mug-deficient E. coli strains.

On the x-axis concentration (mM) of CAA used for modification of plasmids is given, on the

y-axis mutation frequency (MF), expressed as the number of mutants per 104 transformed

cells, is given. Data are expressed as mean ± S.D. of three independent experiments. The

pIF102-C and pIF102-HEC headings of A and B groups of panels, respectively, refer to

plasmids submitted or not to the procedure of transformation of HEC to C (see Section 2.3

for details). The genetic backgrounds of stains are indicated on each panel. The crossed and

filled bars represent MF values obtained in bacteria with and without induction of adaptive

response, respectively. The statistical significance of difference between these groups for each

bacterial strain is denoted by asterisks (* P<0.05, ** P<0.01, *** P<0.001) for MF values

higher, and by hashes (#P<0.05, ## P<0.01, ### P<0.001) for MF values lower in non-

induced than in induced bacteria, respectively.

Fig. 3. Frequencies of GC→TA mutations induced by CAA in the lacZ gene of pIF104

plasmid replicated in wild type and various AlkA-, AlkB- and Mug-deficient E. coli strains.

See legend to Fig. 2 for other explanations.

Fig. 4. HPLC profiles of HEC and C repair by AlkB protein. Repair of HEC: panel A, 5 min

reaction; panel B, 120 min reaction. Repair of C: panel C, 5 min reaction; panel D, 120 min

reaction. The assays were performed at pH 4.6, which was found optimal for repair of both

adducts. See Section 2.9 for other details. Peak appearing in HPLC profile at about 19 min is

dithiotreithol. T/C, T/HEC and T/C denote TTCTT, TT(HEC)TT and TT(C)TT,

respectively.

Fig. 5. The pH-dependence (panel A) and time course (panel B) of repair of m3C, C and

HEC by AlkB protein. Panel A: samples were assayed for extent of repair after 30 min of

reaction at pH values marked in Figure. Panel B: samples were assayed for extent of repair at

pH 4.6 for HEC and C, and at pH 8.0 for m3C. Notice that enzyme concentration used was 8-

23

fold higher in reaction of C and HEC than in reaction of m3C. See Section 2.9 for other

details.

Table 1. Bacterial strains and plasmids.

Strain Relevant Genotype, description Source of derivation Referenceused for mutagenesis tests

AM1 as DH5α but recA+P1 (AM100) DH5α recA+

srl::Tn10 this study

AM2 as AM1 but alkB117::Tn3 carr P1 (BS87) AM1 this studyAM3 as AM1 but mug-1::Knr P1 (MS50) AM1 this study

AM4 as AM1 but alkA1 P1 (MS23) AM1 Tn10 zee3129 his this study

AM5 as AM2 but mug-1::Knr P1 (MS50) AM2 this studyAM6 as AM4 but mug-1::Knr P1 (MS50) AM4 this studyAM7 as AM4 but alkB117::Tn3 carr P1 (BS87) AM4 this studyused for constructions

DH5 α φ80dlacZ ΔM15, recA1,endA1, gyrA96, thi-1, hsdR17 (rk-,mk+), supE44, relA1, deoR, Δ(lacZYA-argF) U169

Central Collection of Strains, Institute of Biochemisty and Biophysics PAS

EC1651 srl::Tn10 recA+ sup3 melCentral Collection of Strains, Institute of Biochemisty and Biophysics PAS

AM100 P90C [araΔ(lacproB)XIII ] [22]DH5α recA+

srl::Tn10 as DH5α but recA+ srl::Tn10 DH5α EC1651 this study

BS87thr-1 ara-14 leuB6 Δ(gpt-proA) 62 lacY1 tsx-33 supE44amber galK2 hisG4 rfbD1 mgl-51 rpsL31 kdg K51 xyl-5 mtl-1 argE3 thi-1 alkB117::Tn3

[23]

MS50 F’ traD36 lacIqΔ(lacZ)M15 proA+B+/thi rpsL (Strr) endA sbcB15 sbcC201 hsdR4 (rK

mK+) Δ(lac-proAB) mug-1::Knr [22]

MS23

thr-1 ara-14 leuB6 Δ(gpt-proA)62 lacY1 tsx-33 supE44amber galK2 hisG4 rfbD1 mgl-51 rpsL31 kdgK51 xyl-5 mtl-1 argE3 thi-1 alkA1 his+[24]

[24]

EC2445araD139 Δ(araF-lac) U169 rpsL150 relA1 deoC1 ptsF25 rbsR flbB5301 trp-1 Tn10 zee3129 his

Central Collection of Strains, Institute of Biochemisty and Biophysics PAS

AM1 Tn10 zee3129 his as AM1 but Tn10 zee3129 his P1 (EC2445)

AM1 this study

plasmids

pIF 102Derivative of low copy pLDR10 vector, cat (fragment containing the entire lac ZIYA operon from strain CC102 [17] was inserted in the plasmid.

[16]

pIF 104Derivative of low copy pLDR10 vector, cat (fragment containing the entire lac ZIYA operon from strain CC104 [17] was inserted in the plasmid.

[16]

24

25