Role of Nucleotide Excision Repair in Processing of 04-Alkylthymines ...

Vol. 269., No. 41, Issue of‘October 14, pp. 25521-25528,1994 Printed in U.S.A.

THEJ o m a OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of Nucleotide Excision Repairin Processing of 04-Alkylthymines in Human Cells* (Received for publication, May 4, 1994)

Johanna C. Klein, Maria J. Bleeker, HarlofC. P. F. RoelenS, Joseph A. RaffertyO, Geoff P. MargisonO, Humphrey F. BruggheS, Hans vanden ElstS, Gi~k A. van der MarelS, Jacques H. van Boom*,Erik Kriek, and AntonJ. M. Bernsnll From the Division of Molecular Carcinogenesis and the Wivision of Molecular Genetics of the Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands, SGorlaeus Laboratories, State University, P. 0. Box 9502, 2300 RA Leiden, The Netherlands, and the SCRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital NHS Bust, Wilmslow Road, Manchester M20 9BX, United Kingdom

04-Alkylthymines have been implicated as potential was obtained that the miscoding potential of these residues carcinogenic DNAlesions.We have studied the effects of might be very high (up to 80-90%) (4-6). During DNA replica04-methylthymine,04-ethylthymine,and04-n-propyltion in HeLa cells, however, the frequency of mutations induced thymine in a model system in whicha single lesion was by 04-ethythymine (04-EtT) appears to be much lower (-20% located at a defined position on a SV40-based shuttle overall or -40% per adducted strand) (7). Other studiesshowed vector and have found large differences in the effectsthat ofin mammalian cells the persistence of 04-AlkT lesions these lesions in repair-proficient and nucleotide excivaries with the size of the alkyl group (8-10); the half-life of sion repair-deficient cells. In repair-competent human 04-methylthymine (04-MeT) in DNA of cultured cells or rat XP-A ( 2 0 5 )revertant liver varies from 2 t o 20 h, whereas 04-EtT has a half-life of HeLa cells, normal fibroblasts, and cells, all 3 residues were highly mutagenic; a mutation 2-20 days. The processing of these lesions thus appears to 0 % was found for both 04-methylthymine frequency of4 depend on the size of the alkyl group (11).These observations and 04-ethylthymine, whereas that of 04-n-propylthymindicate that 04-AlkT residues areactively repaired in mamine was 4 2 % . These frequencies were independent of theactivity ofthe06-alkylguanine DNA alkyltrans- malian cells. This repair could be mediated by 06-alkylguanine DNA alferase.All three 04-alkylthymines induced T * C transikyltransferases, nucleotide excision repair, or an as yet untions exclusively. In nucleotide excision repair-deficient XP-A cells, however, these lesions were not mutagenic known mechanism (4,9, 12, 13). In Escherichia coli 04-AlkT (>go%). These lesions are substratesfor the ada- and ogt-encoded alkyltransbut strongly inhibited plasmid replication ferases that normally act on 06-alkyldeoxyguanosine residues resultsindicatethat04-alkylthyminesareefficiently in DNA (9, 14, 15). 04-EtT can also be removed by nucleotide recognized by the nucleotide excision repair system and excision repair if alkyltransferases arenot inducedand there is cause a complete cessation of plasmid replication if this virtually no repair of these lesions in bacterial cells that lack systemisdeficient.Nevertheless,proficiencyinthe nucleotide excision repair pathway correlates with a both the alkyltransferases and the nucleotide excision repair high frequency of mutation induction by these lesions. system (16). The mammalian alkyltransferase efficiently removes alkylgroups from the 0-6of dG, and, while it was thought that this 17, 18, Carcinogenic alkylating agents induce a variety of aberrant protein could not or could very poorly repair 04-AlkT (9, structures upon reaction with DNA. These DNA lesions (N- or 19),one study reported thepresence of low levels of a 04-MeT0-adducted bases or 0-alkylphosphate lesions) may have dif- specific transferase-like activity in human liver (12), whereas ferent mutagenic and carcinogenic properties, depending on another suggested that the removal of 04-EtT residuesoccurs the position and size of the alkyl group, which can be modu- by a non-transferase mode of repair (13).Recently it was shown bind to doublelated by the presence of specific repair enzymes in a certain cell that the yeast and human alkyltransferase can stranded oligodeoxynucleotides that contain 04-MeT,although type and the DNA sequence context of the lesion (1-4). Alkyl adducts at 0-4 of thymine (04-AlkT)’have been impli- with a low affinity (20). Evidence for the actual removal of now emerging (21, cated as highly mutagenic lesions that cause transitions by 04-MeTby both of these alkyltransferases is mispairing during replication (5). From experiments using al- 22). In mammalian cells, it might also be that alkylatednuclekylated DNA and DNA polymerases in vitro, indirect evidence otides are repaired by nucleotide excision repair. Evidence for the removal of 06-alkyldeoxyguanosine by this repair mechanism in human cells has been published (23-25). * This work was supported by Grant NKI 87-17 from the Dutch CanIn the present study, we investigated whether 04-AlkTs in cer Society, NKB, and a grant from the Cancer Research Campaign. The human cells are substrate for nucleotide excision repair by costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- comparing the mutagenic effects of individual 04-MeT, 04-EtT, tisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate and 04-nPrTmoieties during replication in HeLa cells, normal this fact. 11 To whom correspondence should be addressed. %I.: 31-20-5121991; (SV40-transformed) human fibroblasts and excision repair-deficient xeroderma pigmentosumcells of complementation group Fax: 31-20-5122011. ’ The abbreviations used are: 04-AlkT, 04-alkylthymine; 0‘-EtT, 04- A (XP-A). All residues were located at a single position of a ethylthymine; 04-MeT, 04-methylthymine;04-nPrT,04-n-propylthym- SV40-based plasmid that was transfected into thevarious cell ine; 06-MedG, 06-methyldeoxyguanosine; dG-C8-AAF, N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene; N-acetoxy-AAF, N-acetoxy-N- lines and allowed to replicate transiently. We also determined acetyl-2-aminofluorenedeoxyguanosine; HPLC, high performance liq- the mutagenicity of 06-MedG in relation to theactivity of the uid chromatography. alkyltransferase in the same setof cell lines. Our experiments

25521

25522

Repair Mutagenicity and

of O4-A1kylTin Human Cells

provide evidence that the mutagenicityof 04-AlkTs in human cells is not influenced by the alkyltransferase activity but is primarily caused by incomplete excision repair.

1

S

.*!

-G$ATTCATCGATATCT&GATCT

-

t - 4 0 O b p "$ -+I

"

SV40 Ori

3 __ CTTAAGTBTAGATCTAGA " i f MATERIALSANDMETHODS Enzymes a n d Cells-All enzymes used were purchased from Boehringer Mannheim. FIG. 1.Schematic representation of the position of the 04-alHeLa cells were obtainedfrom the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium supplemented with kylthymines (marked with 0 ) and the modified (OB-Me-or C88% fetal calf serum. The SV40-transformed excision repair-proficient AAF-)dG (marked with *) with respect to the origin of replicahuman fibroblasts (SV40 wild type Amsterdam, referred to as WtA tion on plasmid pSVsupF. These modified nucleotideswereall located in a single-stranded oligodeoxynucleotide (5'-AATTCATCfibroblasts) and SV40-transformed XP-A cell lines(xP2OS-SVand XP12RO-SV) were a gift from Dr. J. H. J. Hoeijmakers (Erasmus Uni- GATATCTA-3', broken underline) which was inserted into gapped duversity, Rotterdam). TheXP2OS-SV revertant cells (clone A24) were a plex forms of the plasmid. Within the oligomer sequence, the modified residues wereplaced at the unique ClaI site (solid underline). The ClaI gift from Dr. P. B. G. M. Belt (Veterinary Institute Lelystad, The Nethcleavage sites are indicated by vertical arrows. The leading and lagging erlands). These cells were cultured inDulbecco's modified Eagle's me- strand during replication are indicated with t and +, respectively. dium with 10% fetal calf serum. Hanahan-competent E. coli DH5a cells (transformation efficiency 108-109/pg) wereobtainedfrom Life Technologies, Inc. and used as directly transformed into Hanahan-competent E. coli DH5a cells, and a n equal portion was additionally cleaved by ClaI before transformarecommended. tion. The mutation frequency was calculated from the relative numbers Plasmids-The plasmids used were described earlier (7, 26); both contained the complete SV40 early region and origin of replication. of transformants obtained mutation frequency = number of colonies pSVsupF carried the alkylated nucleotide at the unique ClaI site and obtained from ClaI + DpnI digested matenallnumber of colonies obtained from DpnI-digestedmaterial. Mutant DNAs werereisolated contained the p-lactamase (Amp') gene for selection in bacteria. Plasfrom E. coli colonies and analyzed using restriction enzyme digestions mid pSVctr served as internalunmodified control and carried the bacand dideoxy sequencing on double-stranded plasmid DNA (7, 31). Per terial chloramphenicol resistance (Cm') gene as selection marker. lesion and per cell type, 2 4 0 clones were analyzedby restriction fragSynthesis and Analysis of 0-Alkylated OligodeoxynucleotidesUnmodified oligonucleotides (5'-dAATTCATCGATATCTA-3'), 04-AlkT, ment analysis, and,of these, 2-10 were sequenced. and 06-MedG containing oligomers (5'-dAATTCA(04-EtT)CGATA- Replication Impairmentof Adducted Plasmids-In all transfections, the unmodified pSVctr plasmid was cotransfected as internal control. TCTA-3' and 5'-dAATTCATC(08-MeG)ATATCTA-3') werechemically Since both plasmids carried different selection markers, the replication synthesized using thesolid phase phosphoamidite method describedby Roelen et al. (27, 28). The oligomer with both an 04-EtT- and a N-(de- inhibition of adduct-carrying plasmidscould be measured from the relative numbers of Amp' versus Cm' E. coli colonies obtained after transoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (dG-CS-AAF) residue was formation of DpnI-cleaved material: relative replication = (number of obtained by reacting the purified ethylated oligodeoxynucleotide with N-acetoxy-AAF as described previously (26). All oligodeoxynucleotides Amp' colonies/pg of DNA transfected)/(number of Cm' coloniesipg of DNA transfected). We also determined the ratios ofAmp'E. coli colonies were extensively purified and analyzed on a Pharmacia PepRPC reversed phase fast protein liquid chromatography column using a buffer obtained from two parallel transfections withmodified and unmanipulated pSVsupF. Average values were calculatedfrom at least two indeof 0.1 M triethylammonium acetate (pH6.9-7.0) with a linear gradient of 10-35% acetonitrile during 45 min. Additionally, the oligomers were pendent sets of experiments. Alkyltransferase Activity Assay-Determination of the alkyltransenzymatically hydrolyzed to composing deoxynucleosides using venom phosphodiesterase (1milliuniupg), nucleasePl(0.05 unitdpg), alkaline ferase activity in the different cell lines was performed essentially as described elsewhere (32,33). Cells were trypsinized, washed phoswith phosphatase (0.05 units/pg) and DNase I (0.05 unitdpg) ina buffer of 50 mM bis-Tris (pH7.2), 2 mM magnesium chloride, and 1mM zinc chloride phate-buffered saline, and pelleted by Centrifugation for 5 min at 1500 rpm. Cells were resuspended (2 x lo6 cells/ml) in a buffer of 50 mM (7). Hydrolysates were analyzed by reversed phase HPLC using a SuTris-HC1 (pH 8.3), 1 mM EDTA, 3 mM dithiothreitol containing 5pdml pelco LC 18s column and a buffer of 40 mM ammonium acetate (pH 4.0) leupeptin and disrupted by sonication (10s at 10p followed by 10 s at with gradients of 5-30% acetonitrile during the first 30 min and 3016 p) after which point phenylmethylsulfonyl fluoride (87 pg/ml) was 45% acetonitrile during the next10 min (see Fig. 2). added. Cellular debris was removed by centrifugation for 10 min at Construction of Site-specificModified Plasmids-Per construct, 250 13,000 rpmat 4 "C. Allquots of the supernatantswere incubatedfor 1h pg of plasmid DNA was digested with EcoRI and BglII in order to at 37"C with calf thymus DNA(-14.5 Cilmmol) that hadbeen methylremove the endogenoustargetsequenceand mixed with an equal ated by reaction with [3H]MNU.Specific activities were calculated from amount of BamHI-linearized plasmid t o a concentration of 20 pg of the fmol of methyL3H transferred to normalized amounts of protein. DNNml in a buffer containing 50% (v/v) deionized formamide, 50 mM Values represent the meanof five independent determinations. Tris-HC1 (pH 7.5),2 mM EDTA, 0.3 M sodium acetate. This sample was denatured by heating for 10 min at 85 "C and allowed to reannealslowly RESULTS by cooling from 60 "C to room temperature. The DNA was precipated with ethanol, and the gapped duplexes were separated from the linear Analysis of Site-specific Modified Plasmids-Constructs with starting material by electrophoresis on agarose gels and subsequent an 04-AlkT-, an 06-MedG, or both an 04-EtTplus dG-C8-AAF' electroelution (7). of residue at the unique ClaI site were obtained after ligation Purified single-stranded oligodeoxynucleotides were phosphorylated chemically synthesized oligodeoxynucleotides carrying justone and ligated into the gapped plasmids as described (7). The resulting of the modified nucleotides into plasmidmolecules that contain covalently closed constructs were separated from remaining nicked a gap at the desired region (see "Materials and Methods"). A plasmids on 4 ml of CsCl density gradients. All constructs were anaschematic representation of the region of modification of the lyzed for the presence of the various lesions at the appropriate siteby restriction enzyme cleavage. constructs is given in Fig. 1. Mutagenicity Assay-Cells were seeded into 60-mm (diameter) Petri Before ligation, alloligomers were extensively purified using dishes and transfected 24 h later at approximately 50% confluence reversed phase fast protein liquid chromatography. Levels of using the calcium phosphate coprecipitation technique of Chen and Okayama (29). Per dish, 6 pg of DNA was transfected consisting of 1pg contaminations werebelow detection limits (51%)with analytical fast protein liquid chromatography (Fig. 2a). The modifiof unmodified or adducted pSVsupF, 1 pg of pSVctr, and 4 pg ofPACYC184 as carrier. The precipitate was left on the cells for12-14 h. The cation of the oligomers is also demonstrated by the altered cells were washed twice, trypsinized after 8-24 h of additional growth retention times (Fig. 2a). The oligodeoxynucleotides were enand divided over two new dishes. Cells were lysed after 68 h, and the zymatically hydrolyzed to nucleosides and analyzed with replasmid DNA was reisolated using a modification of the Hirt extraction versed phase HPLC. Examples of these analysis are shown in procedure (7,30). Thecomplete DNA isolate was digested with DpnI in order to remove unreplicated input material; DpnI specifically cuts Fig. 2b. As was already described by Roelen et al. (27, 28) all DNA that carries the E. coli dam methylation pattern. This pattern is oligomers contained the correct relative amounts of the four lost after replication in eukaryotic cells. One-tenth of this material was regular nucleosides and incase of modification, just one modi-

-

25523

Repair and Mutagenicity of O4-Alky1T in Human Cells

C

B

A

0.2

,i

0.1

wt

dC

0.5

0

04MeT

1.5

1

L ,L 0.2

0.l

0.5

0

0

1

0.2

0.1 0.5

0

1.5 1 0.5

0

0

I

0

20

40

Oc?EtT

04.EIT

L 04-nPrT

06-MedG

1

0

.

1

20

1

1

40

1

-

240

320 200

-

time (min) wavelength (nm) FIG.2. a,reversed phase fast protein liquid chromatography analysis of purified unmodified and modified oligodeoxynucleotides;b HPLC profiles of enzyme hydrolyzed oligodeoxynucleotides. These carried the wild type undamaged sequence (wt)or contained a single 04-AlkT or an 06-MedG. Samples contained the correct relative amounts of nucleosides as was determined in previous experiments (27, 28). See “Materials and Methods” for experimental details. c, UV absorption spectra of the modified nucleosides.

25524

Repair and Mutagenicity of 04-AlkylT in HumanCells TABLEI

Mutation frequencies found after replication of different04-Talkylated pSVsupF plasmids in humancell lines with different repair capacities The single 04-AlkTresidue was locatedat the unique ClaI site of the plasmids. These damaged plasmids and controls were transfected into HeLa cells, normal fibroblasts, and nucleotide excisionrepair deficient XP-A cells, allowed toreplicate transiently, and reisolated. Mutation frequencies were calculated from the fraction of ClaI resistant plasmids among the total number of progeny clones. Construct

Cell type

Mean mutation frequency f S.E.

Wild type plasmid”

HeLa Fibroblasts*

0.22 0.06 1 2 0.3 1 i: 0.3 2 i: 0.3 2 i: 0.8 2 2 0.8 17 5 0 19 i: 2 -0 21 i: 2 18 2 3 121 11 i: 3 12 2 2 =O

No. of experiments

Mean no. of progeny plasmid clonesfpg DNA * S.E.

Total no. of mutants

%

+Unmod. 01.‘ +04-MeT +04-EtT

+04-~m

a

XP-A (20s) HeLa Fibroblasts* XP-A (20s) HeLa Fibroblasts* XP-A (20s) HeLa Fibroblasts* XP-A (20s) HeLa Fibroblastsb XP-A (20s)

5 4 5 4 5 5 2 2 2 9 6 6 7 7 4

6 31 310032 9500 69 13 7 7700 460 150 0 890 550 2 420 250 0

5500 2 1700 5800 2 2300 2 1100 2 1100 3900 2 1600 2500 2 600 2 2800 3700 -c 870 240 2 140 7300 2 1800 3600 2 1300 160 2 60 6400 & 1600 2400 2 710 220 2 80

Unmanipulated plasmid.

* Normal WtA fibroblasts

+ unmod. ol., constructs containing an unmodified chemicallysynthesized oligonucleotide.

fied T or dG (27, 28). Deviations from the correct values were maximally 3.7% and, on average, 2.3% (27,28). These are due to experimental variations in thehydrolysis and chromatography steps. The modified nucleosides had the appropriate W absorption characteristics (Fig. 2c) and comigrated with coinjected reference nucleosides. The 04-EtT containing oligomer sample wasalso analyzed by 32Ppostlabeling in previous studies (7) and appeared to contain less than1% impurities. Purifiedadductedplasmids were subjected torestriction fragment analysis. Plasmids carryinga lesion at the CZaI site could not be cleaved by CZaI, whereas unmodified controls could. Enzymes recognizing adjacent sequences cleaved both unmodified and adducted plasmids. Mutagenicity of Individual 04-AZkT Lesions in Human Cells-Unmanipulated (wild type) plasmids, controls obtained after insertion of an unmodified oligomer or site-specific modified plasmids were transfected into the different repair-proficient and repair-deficient human cells. Plasmids were allowed to replicate transiently and were reisolated; unreplicated material wasexcluded from further analysis. Replicated plasmids were analyzed for the presence of mutations at the original region of modification (CZaI restriction site) and mutationfrequencies were established from the relative numbers of mutated plasmids among the total amount of progeny (see “Materials and Methods”). Table I shows the mutation frequencies found after replication of the different constructs in the chosen set of cell lines. Except for the clones derived from wild type plasmids, we verified the presence of a mutation on a large number of clones by means of restriction fragment analysis. The reliability of the CZaI selection was also verified by the fact that cotransfected control plasmids (also carrying a CZaI site) were completely linearized. Progeny of untreated plasmids isolated from all cell lines carried (less than) 0.2-1% mutations. These clones were not analyzed further. Insertion of an unmodified oligomer slightly increased the mutation frequency to -2%. This frequency was found with all cell lines tested. Table I1 shows the spectrum and distribution of the mutations found after sequencing of the mutant clones. The mutations, single base pair substitutions or small deletions, appeared to be randomly distributed over the region of modification. In addition, multiple mutations were found with

TABLE I1 Specificity and frequency of mutations present in mutantclones derived from pSVsupF plasmids with site-specific 04-AlkTresidues after replication in different human cell lines The individual 04-AlkTswere inserted into these plasmids by ligation with chemically synthesized oligomers having the sequence 5“AATTCA(04-AlkT)CGATATCTA-3’ (see “Materials and Methods”). Control constructs carried an unmodified oligomer at the same region. Shown are theabsolute frequencies of the three different groups of mutations found. Mutations +

construct

HeLa + unmodified oligonucleotide HeLa + 04-MeT HeLa + 04-EtT” HeLa + 04-nPrT

Targeted Other point Multiple T+ mutations C mutations %

%

%

0.2

1

0.8

17 20 10

0 0 0

0 1 1

Fibroblast’ + unmodified oligonucleotide Fibroblastb + 04-MeT Fibroblast’ + 04-EtT Fibroblast* + 04-nPrT

0

2

0

19

0

18 12

0 0

0 0 0

XP-A (20S)+04-EtT

1

0

0

Values forthe mutational specificityof 04-EtTin HeLa cells were in previous studies: 21% T to C transitions, 1% other point mutations, and 2% multiple mutations (7).The multiple mutations consisted of a number of single base pair substitutions combined with small deletions as described previously (7, 26). bNormal WtA fibroblasts.

a characteristic pattern consisting of base pair substitutions with small deletions. These mutations have also been found in previous studies (7). The presence of an 04-MeTor an 04-EtTincreased the mutation frequency t o -20% among progeny plasmids derived from repair proficient HeLa cells and WtA fibroblasts. Almost all mutationswere found at theposition of the alkylatedT and consisted of a substitution into dC. The remainder of the mutations were multiple mutations of the type described above (see Table 11). The mutation frequency induced by 04-nPrT in thetwo repair proficient cell lines was approximately halfof that induced

Repair Mutagenicity and

of O4-Alky1T Human inCells

25525

TABLE I11 Mutation frequencies among progeny of pSVsupF plasmids carrying individual 04-EtT residues after transfection into different XP-A and XP-A revertant cell lines These frequencies werealso determined for undamaged (Wt) plasmids. Values were calculated from the relative numbersof progeny plasmids harboring an sequence alteration at the region of modification COnStNCt

Cell type

Mean mutation frequency 2 S.E.

Wt plasmid

XP-20s XP-20s rev XP-12RO XP-20s XP-20s rev XP-12R0

1 f 0.3 f 0.3 0.2 f 0.1 1f1 24 f 12 25 f 5

No. of experiments

Total no. of mutants

5 3

32

4 6 2 4

1

clones Ipg DNA

%

0.5

+04-EtT

2

Mean no. of pro eny plasmid f S . g .

2

11 34

3100 f 1100 1300 f 900 3000 f 1200 160 f 60 400 f 150 590 f 220

by 04-MeT and 04-EtT (on average 12 versus 20%)(Table I). In the smallest lacksexon 3 entirely and carriesa new stop codon stop codon in exon all sets of parallel transfections, we reproducibly found lower in exon 4, the largestonly contains the extra mutation frequencies for 04-nPrT thanfor 04-MeT or 04-EtT, 4 (34, 35). Both mRNAs yield largely truncated XP-A factors. and for the mutation frequencies of 04-EtT and 04-nPrT in The XP20S revertant cells have not been characterized at a HeLa cells, the difference was statistically significant ( p < molecular level, but their UV survival capacity is nearly equal 0.02). 0 4 - n P f l also exclusively induced T C transitions. The to wild type levels (36). XPl2RO cells contain severely reduced few other mutationsfound consisted of the multiple mutations amounts of normal sized D A C mRNA. However, this RNA that were alsoobserved with the analysis of controls containing contains a nonsense mutation in thefifth exon, giving rise to a truncated (207-amino acid) XP-A protein that lacks the 67 Cunmodified oligomers (Table 11). We could not determine the mutagenicity of 04-AlkT in XP-A terminal amino acids (34, 35). The replication of wild type and 04-EtT-adductedpSVsupF cells, since all 3 residues extensively inhibited plasmid replication (see below). The few replicated clones that were isolated plasmids relative t o the replication of cotransfected pSVctr concarried almost exclusively the wild type sequence. From trols inall cell lines isshown in Fig. 4. The relative amounts of 04-EtT-adductedplasmids, only two clones were obtained that the two unmodified plasmids undergoing replication appear t o C transition. However, since plasmid be cell line-specific. Similar ratios were found for adducted carried the specific T replication was so severely impaired, it is not clear whether plasmids replicating in excision repair-proficient HeLa cells, fibroblasts, and X P 2 0 S revertant cells. Adducted plasmids these two mutants were specifically induced by 04-EtT. Interference with Plasmid Replication in XP20S (XP-A) failed to replicate only in the XPBOS-SV cell line (ratio 0.01) Cells-In order to measure selective loss of the plasmids due to and were very inefficiently replicated in theXP12RO-SV cells replication inhibition by the modifications, we calculated the (ratio 0.06) in comparison t o undamaged plasmids, which proratios for the (normalized) amounts of progeny derived from duced ratios of 0.25 and 0.98, respectively. This implies that adducted pSVsupF plasmids and the corresponding unmanipu- in XP12RO cells a relatively larger fraction of adducted plasmids has replicated. lated pSVsupF controls in parallel transfections. These The mutation frequency of 04-EtT in XPl2RO was -25% amounts were determined using E. coli transformations (as is described under "Materials and Methods"). Mean ratios from at (Table 111).Mutations were again T + C transitions. Inx P 2 0 S least two independent sets of experiments aredepicted in Fig. revertant cells, the mutation frequency of 04-EtT was 24%, which is comparable with the frequency found for the other 3. In addition, adducted plasmids were always cotransfected with equal amountsof an unmodified control plasmid (pSVctr). repair proficient cell types. The majority (-21%) of the mutaC transitions. We also determined the ratios relative to the amount of progeny tions were T Comparison with the Mutagenicity of 06-MedG-To exclude a derived from pSVctr control plasmids (Fig. 4). In both of the excision repair-proficient cell lines, adducted possible influence of the alkyltransferase on the mutagenicity constructs andcontrols carrying anunmodified oligonucleotide of 04-AlkT,we transfected plasmids carrying a single 06-MedG yielded approximately equal amounts of replicated plasmids at the ClaI site into the same cell lines. As is shown in Table IV, (ratios were -0.9 for HeLa and -0.7 for WtA fibroblasts, Fig. the mutationfrequency of 06-MedG varied considerably among 3). When ratios were established relative t o the replication of the different cell types. The mutagenicity in HeLa cells was cotransfected pSVctr plasmids in these cells, we also found comparable with that of 04-MeT and 04-EtT (mutation fresimilar values (-0.4 for HeLa and -0.5 for WtA fibroblasts). quencies of 14 and 17-21%, respectively), whereas in WtA fiHowever, in XP20S cells, plasmids carrying either one of the broblasts and XPPOS cells, the mutation frequency (2-5%) 04-AlkTlesions wereless well replicated relative toboth of the hardly exceeded background levels (2%).The majority of the controls (undamaged pSVsupF and cotransfected pSVctr); the mutations consisted of a G + A transition at the former posiamount of progeny obtained from adducted constructs was only tion of the adducted dG (Table V), which is in good agree5-8% (ratios of 0.05-0.08) of that obtained from wild type plas- ment with the mutational specificity of 06-MedG reported in mids transfected in parallel experiments, while controls cany- literature (17). ing an unmodified oligodeoxynucleotide replicated almost as The specific activity of the alkyltransferase varied from 18 well as the untreated plasmids (Fig. 3). fmol/mg of protein for the XP20S cells t o 768 fmoVmg ofprotein Effects of 04-EtTin Other XP-A cells and a XP-A Revertant for the WtA fibroblasts (see Table IV). The mutation frequenCell Line-To ascertainthatthe replicationinhibition in cies were in agreement with the alkyltransferase activity in the X P 2 0 S cells was indeedcaused by the nonfunctional XP-A two excision repair proficient cell lines; the high mutation frefactor, we introduced constructs with an 04-EtT into another quency of 06-MedG in HeLa cells corresponds with the relaXP-A cell line (XP12RO-SV) and revertant cells (clone A24) tively lower alkyltransferase activity in these cells and the from the XP2OS-SV line. much lower mutation frequency in WtA fibroblasts with the X P 2 0 S cells are homozygous for a splicing mutation in in- higher alkyltransferase activity. tron 3, which results intwo abnormally spliced XPAC mRNAs; The low mutation frequency of 06-MedG in x P 2 0 S cells does "-f

--f

"-f

25526

Repair and Mutagenicity of O4-Alky1T in Human Cells

FIG.3. Replication of pSVsupFplasmids containing a single 04-AlkTor an unmodified oligonucleotide (unm) as compared with the replication of unmanipulatedpSVsupF in different human cell lines. Adducted plasmids and controls were transfected in parallel experiments. Normalized amounts of progeny plasmids were determinedfor each transfection from thenumber of Amp' clones. Solid bars represent mean ratios from threeindependentexperiments; error bars represent the S.E. The different cell lines are indicated with H (HeLa),F (normal fibroblasts, SV40 Wt Amsterdam), X l r (XPBOS-SV revertant clone A24), and X 1 (XP-Acell line XPBOSSV). See text for experimental details.

H F X1

H F

xi

H F X1 Xlr cell line

H F X1 --c

DISCUSSION

We have determined the mutagenicity of 04-MeT, 04-EtT, + &tT and 04-nPrT residues in humancells with different repair capacities using site-specific modified plasmids. All three lesions were highly mutagenicin excision repair-proficient cells (-20% for methyl and ethyl adducts and -12% for n-propyl adducts) 1 and specifically induced T + C transitions. The mutational specificity of the 04-AlkT residues is asexpected and in accordance with our previous studies on the mutagenicity of 04-EtT inHeLa cells (7). The same transitions were also found by other groups investigating the effects of 04-MeT and 04-EtT during replication in E. coli and inin vitro assays (5, 37-39). The mutation frequencies induced by these lesions in our system, however, were lower than those calcuH F X1 XlrX2 H F X1 XlrX2 lated from in vitro experiments (4, 6). This suggests that there is repair of 04-AlkTs in human cells, although this may be inefficient. cell line In order to investigate what type(s) of repair could be inFIG.4. Relative replication of unmanipulated (- -) or 04-EtT adducted pSVsupF plasmids as compared with cotransfected volved, we used different cell lines that varied largely with unmodifiedpSVctr controls in different human cell lines. This is respect to the repair activities of the 06-alkylguanine DNA expressed as normalized amounts ofAmp' uersus Cm' clones. Solid bars alkyltransferase or nucleotide excision repair system (AT+/ represent the average ratios from at least twoindependent experiments; error bars represent the maximal variation observed between NER', AT-/NER+,AT-/NER-). In E. coli, 04-AlkTs are primarily individual determinations.The different cell lines are indicated with H removed by the 06-alkylguanine DNA alkyltransferase (9, 15, (HeLa),F (normal fibroblasts, SV40 Wt Amsterdam), X1 (XP-A line 331, but it appearsunlikely that this is the case in humancells. XPBOS-SV), X l r (XPBOS-SV revertant clone A241, and X2 (XP-A line In our studies, alkyltransferase activities varied widely in the XPlPRO-SV). See "Materials and Methods" for experimental details. different cell lines and correlated well with the mutation frequency of 06-MedG, but not at all with that of 04-AlkT. The not correlate with the very low alkyltransferase activity in alkyltransferase activity in HeLa cells appeared relatively low these cells, since adducted plasmids again almost completely as compared with that inWtA fibroblasts, and this isreflected failed to replicate. Only two mutant clones were obtained both in the much higher mutation frequency of 06-MedG in HeLa of which carried the G + A transition at the position of the cells. However, mutation frequencies of each of the 04-AlkT modified dG, but again it is not clear whether these two mu- lesions weresimilar inboth HeLacells and WtA fibroblasts and approximately equal t o those reported for 06-MedG (04-Me-, tants were spontaneously or specifically induced. 04-EtT) and 06-EtdG (04-nPrT) in alkyltransferase-deficient MutationInductionin Repair-competent Cells-The observed loss of adducted plasmids in XP-A cells indicates that CHO cells (17). It has been reported recently that the human alkyltrans04-AlkT and 06-MedG residues are recognized by nucleotide excision repair. Since the lesions are still highly mutagenic in ferase is able to bind with very low affinity to 04-MeTin vitro repair competent cells, we hypothesized that in thesecells ex- but notto 04-EtT(20). Hence, it wassuggested that the human to extent in vivo cision of the damaged nucleotidesis incomplete, possibly due to alkyltransferase may alsorepair 04-MeT some the relatively small size of 04-AlkT residues. To test this hy- (20), and this may explain the differences in biological halfchromosomal DNA as observed pothesis, we constructed plasmids with an 04-EtT and a dG- lives of 04-MeT and 04-EtT in with other experimental systems(tl,2of 2-20 h and 2-20 days, C8-AAF next toeach other in the same strand. dG-CS-AAF The was located 2 base pairs3' to the 04-EtT (see Fig. 1).When this respectively) (8-10). Such repair might have influenced the mutation frequency of 04-MeT inour system as well, but this construct was transfected into the fibroblasts, the mutation frequency did not exceed the background frequency (-2%) and frequency appeared to be similar to the mutationfrequency of was comparable with that of dG-C8-AAF alone (Table VI; Ref. 04-EtT (-20%). Therefore, alkyltransferases do not seem to 26). The mutations were of the same types asfound with con- play a prominent role in theremoval of any of the 04-AlkTs in human cells. trol constructs.

-

25527

Repair and Mutagenicity of O4-A1ky1Tin Human Cells

TABLE IV Mutation frequencies found after replicationof pSVsupF plasmids withsite-specific 06-MedG residues in different human cell lines These frequencies are related to the specific activities of the 06-alkylguanineDNA alkyltransferase (ATase).The 06-MedGresidues were located at the unique ClaI site of the plasmid. Mutation frequencies were determined from the relative numbers of progeny plasmids that were resistant to cleavage with ClaI. Cell type frequency

Mean mutation S,E. ~

No. of experiments Total

no. of mutants

7c

HeLa Fibroblast" XP-A (20s) a

14 t 4 5+_3 222

2

2

22 9

2

2

Mutations point Targeted Other typeCell

Multiple G-A

HeLa Fibroblastsb XF"A ( 2 0 s )

mutations" mutations

%

%

%

14 3 2

0 1 0

0

1 0

The multiple mutations have been described elsewhere ( 7 , 2 6 ) . Normal WtA fibroblasts.

VI TABLE Mutation frequencies induced by a single 04-EtT a dG-C8-AAF or a double modification with anO4 EtT and a dG-Cg-AAFnext to each other in the same strand of pSVsupF plasmids replicating in normal W t A fibroblasts Construct B

oligonucleotide +unmodified +04-EtT +dG-C8-AAF" +O*-EtT+ dG-C8-AAF a

Mean ATase activity S,E,

cloneslpg DNA

fmol methyl-3Himg protein

1500 2 650 4300 3500 390 2 80

347 2 5 768 t 39 18 2 5

~

No. of experiments

5 5 5

Normal WtA fibroblasts.

TABLEV Specificity and frequencyof the mutations found after sequencing of the mutant clones derived from plasmids withsingle 06-MedG residues that were replicated in normal and excision repair deficient human cells Shown are the absolute frequencies of the different types of mutations found.

Mutation

Mean no. of progeny plasmid +S.E.

2 18 2

Data taken from Ref. 26.

Our data indicate that 04-AlkTmoieties are recognized by nucleotide excision repair. Remarkably, we found these lesions to be inhibitory to plasmid replication and, consequently, not to be mutagenic innucleotide excision repair-deficient XP-A cells. The replication inhibition of 04-EtT adducted plasmids was observed with two different XP-A cell lines (XP2OS-SV and XP12RO-SV), both of which are highly deficient in nucleotide excision repair but carrydifferent mutations in theXP-A gene (34, 35, 40, 41). This makes it unlikely that the replication those affectinhibition canbe ascribed t o mutations other than ing theexcision repair capacity. Moreover, the replication block was relieved in a revertant of the XPZOS-SV cell line, and the mutation frequency of 04-EtT in thesecells was equal to that found with the other repair proficient cells. 04-EtT also appeared t o inhibit plasmidreplication in XP-D (HD2) cells. These cells carry a mutation in a different protein involved in nucleotide excision repair (421, and also, inthis case, the amount of progeny derived from ethylated plasmids was largely reduced (-65% as compared with controls). This confirms our notion that the alkylated plasmids indeed fail to replicate because the 04-AlkT lesions are not removed and, therefore, impede the progress of the replication fork. Another indication for the removal of 04-AlkTsby nucleotide excision repair is thefact that mutationfrequencies decreased with increasing size of the adduct (mutation frequency 04-

MeT = 04-EtT > 04-nPrT > 04-EtT + dG-C8-AAF'). If repair was mediated by the alkyltransferase (or a glycosylase), one would expect the mutation frequency to increase with adduct size, as both the E. coli and mammalian alkyltransferases are known to repair larger 0-alkylgroups less efficiently (43, 44, 45). We observed the opposite. The decrease in mutation frequency we found can only be consistent with removal by nucleotide excision repair, since this type of repair has been shown to recognize distortions of the DNA double helix caused by the adducts rather than the adducts themselves (46). With increasing adduct size, the distortion of the helix might become more extensive, allowing a more efficient repair. The effect is most pronounced with dG-C8-AAF, which has been shown to cause large alterations in theDNA conformation and which is indeed e%ciently removed by nucleotide excision repair (26, 47). Finally, it isknown from literature thatdifferent cell lines of the XP-A complementation group failto replicate plasmids carrying other carcinogen-DNA adducts, which have been shown to be substrates for nucleotide excision repair (41, 48, 49). Previous experiments in our laboratory demonstrated that lesions that are substratesfor glycosylase repair (i.e. 8-oxo-dG)do not inhibit DNA replication in XP-A cells (26). From the present studies, it appears that even the smallest 04-AlkT adducts completely prevent 0.5bypass of replication, indicating thatnucleotide excision repair is the predominant repair mode operating on these adducts. The fact that theobserved inhibition of plasmid replication in XP-A cells is (almost) 100% indicates that the alkylated thymines are very efficiently recognized by the nucleotide excision repair system. This is unexpected because from the high mutation frequencies in repair-proficient cells, one would have anticipated a rather poor recognition of 04-AlkTs.Apparently, recognition and incisiodexcision are somehow uncoupled processes, since all lesions (small and large) are recognized effectively but not removed with equalefficiency. It may be that the smaller 04-AlkT lesions do not permit thespecific identification and incision of the damaged strand but that, instead, the undamaged strand is incised, and subsequent repair replication muusing the damaged strandas the template introduces the tation opposite 04-AlkT. Removal of the lesion by the proper operation of the excision repair system might then allow normal plasmid replication t o proceed. Alternatively, the nucleotide excision repair factors in proficient cells might interact with the damage in a way that does not completely suppress replication, whereas in repair-deficient cells this interactionis more extensive and completely blocks replication. The lower mutation frequencies of the larger (04-nPrT and dG-C8-AAF') adducts suggest that the incisiodexcision process is more reliable with larger adducts.When the strandcarrying an 04-EtTwas marked with an additional C8-AAF adduct on a 3' neighboring dG, it might have been predictedthat thiswould reduce the mutagenicity of the adjacent 04-EtT and indeed, simultaneously with theexcision of dG-C8-AAFthe 04-EtT was

25528

Repair and Mutagenicity of O4-A1kylT Human in

removed and the mutationfrequency concomitantly decreased to background levels. The recognition of 04-AlkTsby nucleotide excision repair has also been shown t o occur in E. coli (16). However, Bronstein et al. (25) reported no differences between excision repair deficient and proficient human cells with respect to the t,, values for the removal of 04-AlkT residues from DNA, and they concluded that these lesions were not substrates for nucleotide excision repair. We have not measured the actual removal (t1J of these lesions in oursystem. Our experimentsalso differ with respect to the position of the lesions (plasmid uersus chromosomal DNA), the cell lines used, and their rates of division. Therefore, the apparentdiscrepancy between their conclusions and oursprobably relates togross differences in theexperimental systems that were utilized. Altogether, our experiments demonstrate that04-AlkT residues can be highly mutagenicin humancells, in spiteof the fact that they appear tobe efficiently recognized by the nucleotide repair system. Surprisingly, the nucleotide excision repair system seems instrumental in the induction of mutations at the position of 04-AlkT lesions. This clearly designates these lesions as potential inducers of neoplastic transformation. Acknowledgments-We thank Pim van Dijk for technical assistence with the HPLC analysis and Jos Domen, Heinte Riele, Chris Saris, and Gerard Westra for helpfuland stimulating discussions. We are grateful to Dr. J. Hoeijmakers and Dr. P. Belt for providingus with the X P cell lines and fibroblasts. REFERENCES 1. Burns, P. A., Gordon, A. J. E., Kunsmann, K., and Glickmann, B. W. (1988) Cancer Res. 48, 4455-4458 2. Dolan, M. E., Oplinger, M., and Pegg,A. E. (1988) Carcinogenesis 9,2139-2143 3. Pegg, A. E. (1990)in Chemical Carcinogenesis and MutagenesisI1 (Cooper, C . S., and P. L. Grover, eds) pp. 103-131, Springer-Verlag New York Inc., New York 4. S a f i i l l , R., Margison, G. P., and OConnor, P. J. (1985) Biochim. Biophys. Actu 823,111-145 5. Singer, B. (1986) Cancer Res. 46, 48794885 6. Saffhill, R. (1985) Chem. Biol. Interact. 63, 121-130 7. Klein, J . C., Bleeker, M. J., Lutgerink, J. T., Van Dijk, W. J., Brugghe, H. F., Van den Elst, H., Van der Marel, G. A,, Van Boom, J. H., Westra, J. G., Berns, A. J. M., and Kriek, E. (1990) Nucleic Acids Res. 18, 41314137 8. Bodell, W. J., Singer, B., Thomas, G. H., and Cleaver,J . E. (1979)Nucleic Acids Res. 6,2819-2829 9. Brent, T. P., Dolan, M. E., Fraenkel-Conrat, H., Hall, J. Karran, P., Laval, F., Margison, G. F!, Montesano, R., Pegg, A. E., Potter, P. M., Singer, B., Swenberg J. A,, and Yarosh, D.B. (1988) Proc. Natl. Acad. Sci. U. S . A . 86, 1759-1762 10. DenEngelse, L., Menkveld, G. J., De Brij, R.-J., and Tates, A. D. (1986) Carcinogenesis 7, 393403 11. Richardson, E C., Dyroff, M. C., Boucheron,J. A., and Swenberg, J. A. (1985) Carcinogenesis 6,625-629 12. Becker, R. A,, and Montesano, R. (1985) Carcinogenesis 6,313-317 13. Wani, A. A,, Wani, G., and DAmbrosio, S. M. (1990) Carcinogenesis 11, 14191424

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