analysis of dna alkylation damage and repair in mammalian cells

Mutagenesis vol.11 no.2 pp. 169-175, 19%

Analysis of DNA alkylation damage and repair in mammaliancells by the comet assay

P.Fortini1, G.Raspaglio1, M.Falchi2 and E.Dogliotti1-3

'Laboratory of Comparative Toxicology and Ecotoxicology and 2Laboratoryof Ultrastructures, Istituto Supenore di Saniti, ViaJe Regina Elena 299,00161 Roma, Italy

^To whom correspondence should be addressed

The single cell gel electrophoresis (SCGE) or comet assay,which measures DNA strand breaks in individual cells,was used to analyse DNA damage and repair induced bythe SNrtype alkylating carcinogens N-ethyl-iV'-nitro-iV-nitrosoguanidine and N-ethyl-N-nitrosourea in CHO cells.The comet assay was comparable in sensitivity to thealkaline elution assay. The alkyl-adducts detected as DNAsingle-strand breaks (ssb) by this technique were completelyrepaired within 24 h after treatment. These data indicatethat long-lived lesions, such as alkylphosphotriesters, arenot converted into ssb under the standard SCGE alkalineconditions (pH 13.5). The lesions revealed by the cometassay are mainly apurinic/apyrimidinic (AP) sites andbreaks formed as intermediates in the base excision repairprocess of N-alkylpurines. When SCGE was performed atpH 12.5 instead of pH 13.5 a lower level of ssb was detectedand these breaks were completely resealed within 2 h aftertreatment These data suggest that different subsets oflesions are detected under different pH conditions. TheSCGE combined with inclusion within the cells of endo-nuclease III revealed that a high portion of AP sites inducedby alkylation damage were not converted into ssb by alkali.The level of endonuclease ITi-sensitive sites decreased as afunction of the repair time and by 24 h after treatment nosites were left on the DNA. The use of this modifiedSCGE assay allows the estimation of the total amount ofunrepaired AP sites present on DNA. Alkylation-inducedssb as detected by the comet assay should be regarded asan indicator of repair rate and balance more than ameasure of actual DNA damage.

IntroductionAlkylation damage includes a variety of DNA base modifica-tions which may result in mutations and eventually lead tocarcinogenesis. Living cells counteract these lesions by a setof repair enzymes which specifically recognize these alkylatedbases, often producing sites of base loss.

The relative contribution of DNA abasic sites to themutagenic properties of alkylating agents is still undefined.However, several lines of evidence strongly suggest that abasicsites play an important role in alkylation-induced mutagenesisand cytotoxicity. Endonuclease IV Escherichia coli mutantsand yeast strains deficient in the major apurinic/apyrimidinic(AP) endonuclease APN1, which exhibit a mutator phenotype,are hypermutable by alkylating agents (Cunningham et al.,1986; Ramotar et al., 1991). AP sites induced by alkylationdamage are mainly the result of enzymatic hydrolysis of3-alkyladenine (3-alkylA) and 7-alkylguanine (7-alkylG)

by a DNA N-glycosylase. In agreement with the mutagenicpotential of these AP sites, the overexpression of the3-methyladenine DNA glycosylase (MAG) gene (Chen et al,1989) in a yeast apnl mutant strain increases spontaneousmutation and underexpression decreases spontaneous mutation(Xiao and Samson, 1993). Human cell lines with low levelsof the majoi mammalian AP endonuclease HAP1 are hyper-sensitive to the killing effects of alkylating agents and tooxidative stress (Walker et al., 1994). Moreover, the analysis ofmutational spectra induced by N-nitroso alkylating carcinogens,such as N-ethyl-N-nitrosourea (ENU) and A -̂ethyl-Af'-nitro-A/-nitrosoguanidine (ENNG), in mammalian cells clearly showsthat, besides GC to AT transitions, the second major type ofmutation induced is transversions localized at AT base pairs(Bronstein et al, 1992; Fortini et al, 1993). AP sites are agood candidate for this mutagenic event.

The development of techniques able to measure AP sites istherefore of great interest. The single cell gel electrophoresis(SCGE) or comet assay is a sensitive and rapid method forDNA strand break detection at the level of single cells. A highpH (>13) is generally used in this assay to facilitate DNAdenaturation and unwinding. Under alkaline conditions twotypes of alkylation-induced lesions might become apparent asstrand breaks: abasic sites and alkylphosphotriesters. Bothspontaneous and enzymatic loss of alkylated bases gives riseto AP sites. Spontaneous cleavage at AP sites under conditionssimilar to those present in vivo is a slow process, taking20-100 h, while at alkaline pH (12.8) at 25°C the averagelifetime of an AP site is 32 min (Lindahl and Andersson,1972). Even faster is the enzymatic conversion of AP sitesin vivo by AP endonucleases. By using mammalian cell extractson a depurinated substrate we have estimated that the completerepair of AP sites occurs within 20 min (Frosina et al, 1994).In addition, alkylphosphotriesters, which are quite abundantfollowing DNA alkylation, are hydrolyzed in alkaline condi-tions to single-strand breaks (ssb) with a half-life of 2-3 hat 37°C (Shooter, 1976). Therefore, breaks detected underalkaline conditions should be the sum of true breaks and alkali-labile lesions.

In this paper we have attempted to identify the type of DNAlesions induced by alkylating carcinogens which are detectedas ssb by the comet assay. Moreover, by the combined useof SCGE and inclusion within the cells of bacterial APendonucleases we have exploited the feasibility of measuringthe level of persistent DNA damage as a function of therepair time.

Materials and methods

ChemicalsBoth ENNG and ENU were obtained from Pfaltz and Bauer (Waterbury, CT).These chemicals were dissolved in dimethyl sulfoxide (DMSO) (SigmaChemical Co., St Louis, MO) shortly before use and quickly diluted incell culture medium to the required concentrations (final concentration ofDMSO <0.5%).

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Cell culture and treatmentChinese hamster ovary (CHO)-K1 cells were grown in Ham's F10 medium(Gibco) supplemented with 10% fetal calf serum, 100 U/ml penicillin and100 fig/ml streptomycin. Cells were grown in monolayer culture at 37°C, 5%CO2. CHO cells were irradiated on ice using a Siemens Stabilipan 250 kVX-ray unit at a dose rate of I Gy/min. The irradiation was performed ingrowth medium.

Cells were treated with ENNG or ENU in phosphate-buffered salinesupplemented with 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid (finalconcentration 20 mM) for 30 min at 37°C. Cells were allowed to repair DNAdamage by incubation in fresh medium for the indicated periods of time.

Assay for cell survivalThe cytotoxic effect was determined from the loss of colony-forming ability.CHO cells (100 cells/60 mm dish) were treated as described above, thenwashed, fed with complete medium and 7 days later fixed with methanol andstained with 10% Giemsa. The cloning efficiency of the untreated cells wasclose to 100%.

Single cell gel electrophoresisDNA breaks were detected essentially as described by Gedik et al. (1992).Briefly, cells were embedded in agarose and then spread on a frostedmicroscope slide. Cells were lysed in 2.5 M NaCI, 10 mM Tris-HCI, 100 mMNa2EDTA, 1% Triton, 10% DMSO, pH 10, for 1 h at 4°C. When theendonuclease Ill-sensitive sites were determined, slides were washed threetimes with the enzyme buffer (40 mM HEPES-KOH, 0.1 M KCI, 0.5 mMEDTA, 0.2 mg/ml bovine serum albumin, pH 8.0) and the cell-containingagarose layer was covered with 50 |il °f buffer or endonuclease 111(10 u.g/ml) and incubated for 30 min at 37°C. The purified enzyme was akind gift of Serge Boiteux (Institute Gustave-Roussy, Villejuif, France).Finally, cells were preincubated for 20 min at 4°C in the electrophoresisbuffer (0.3 M NaOH, 1 mM Na2EDTA, pH 13.5) and then subjected toalkaline gel electrophoresis (300 mA, 4°C, 20 min) When indicated, thecomet assay was performed by using an electrophoresis buffer at pH 12 5(0.03 M NaOH, 1 mM Na2EDTA).

Image analysisAfter SCGE, cell DNA was stained with ethidium bromide (2.5 Hg/ml) andvisualized by fluorescence microscopy. Negative photomicrographs wereanalysed by an IBAS 2 image analysis system with 256 grey levels. Greylevels of each image were normalized and background subtractions wereperformed on each normalized image. Different geometric comet featureswere measured on the whole comet, as well as the tail and head regions. Asreported by others (for a review see McKelvey-Martin et al., 1993) the taillength did not always increase as a function of the dose of the test agent. Thetail area, which reflects the displacement of DNA from the head to the tail,was dose-related, and therefore this was taken as the measure of DNA damage.Fifty comets per experimental point were scored.

Results

X-ray-induced DNA damage and repairThe sensitivity of the comet assay under our experimentalconditions was established by exposing the cells to ionizingradiation. The survival curve of CHO cells after exposure toincreasing doses of X-rays is shown in Figure 1 A. DNA breakswere measured in the same dose range by the comet assay(Figure IB). The DNA damage to individual cells is expressedas tail area. The data relative to at least 50 comets per dosepoint are presented as box plots, where the Y axis displays therange of the comet tail area values. This type of plot allowsthe evaluation of the heterogeneity in cell response to DNAdamage. We were able to detect damage at irradiation dosesas low as 2 Gy. At this dose 60% of the cells survived thedamage (Figure 1A).

Ionizing radiation produces a variety of lesions in DNA andthe comet assay should detect mainly single-strand breaks. Ifthis is true the repair kinetics should be extremely fast (Olive,1988). In fact, repair of damage was rapid, with the majorityof breaks resealed after 10 min (Figure 2A). This is inagreement with results reported for V79 cells by Olive et al.(1990). Interestingly, when damaged cells were allowed torepair, a strong reduction of the amount of free DNA piecesthat could migrate (i.e. a strong reduction in the average tail

Xrays(Gy)

B.2000

control 0.5Gy 2Gy 10Gy 20Gy

X rays dose

Fig. 1. X-ray-induced DNA damage in CHO cells. (A) Survival curve. Eachpoint represents the mean of three independent experiments. SD values werealways <10%. (B) DNA breaks as measured by the comet assay. The tailareas of 50 comets per experimental point were measured by imageanalysis. The data are displayed as box plots, where the Y axis shows therange of the data. Each box encloses 50% of the data, with the medianvalue of the variable displayed as a line. The top and the bottom of the boxmark the limits ±25% of the variable population. The lines extending fromthe top and the bottom of each box mark the minimum and maximumvalues that fall within an acceptable range. Any value outside this range isdisplayed as an individual point.

area) was observed shortly after irradiation (10 min repair;Figure 2B). However, the irradiated cell population did notrecover completely the features of the control cells even after30 min repair (Figure 2B). This is likely due to differences inthe packaging of DNA in cells undergoing repair as comparedto unexposed cells. The same phenomenon was observed afteralkylation damage (see below).

If we assume that 1 Gy induces 2.5 ssb per 1010 mol. wt ofDNA, the comet assay has a sensitivity of the order of5 ssb/1010 daltons, i.e. 2000 ssb/cell.

Alkylation-induced DNA damage and repairCHO cells were treated with two SN,-ethylating agents, ENUand ENNG. In a previous study both chemicals were shownto efficiently induce DNA ssb as detected by alkaline elution(Dogliotti et al., 1984). A dose-response curve was observedafter exposure to ENU (Figure 3B) and ENNG (Figure 4B).The comet assay was able to detect damage at doses thatinduced 90% survival (Figures 3A and 4A). When the datawere compared with those obtained with different methods forssb detection, such as alkaline sucrose gradient centrifugationand alkaline elution (data from Dogliotti et al., 1984), the

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DNA damage and repair by the comet assay

A. A.1000

control 5Gy 2 min 10 min 30 min

B.

? f s ?5 » s

TdArM TtlAraa

i "1

10 min repair

1i m

I 30 mh r«p«lr |

? ! ? !S « 8 8

TdAru

? ? ? !s » s 8

TwIAra*

Fig. 2. Repair of X-ray-induced DNA damage in CHO cells. (A) Cells wereirradiated with 5 Gy and then incubated at 37°C. At different time intervalssamples were taken for SCGE. The tail areas of 50 comets per experimentalpoint were measured by image analysis. The data are displayed as box plots(for details see the legend to Figure 1). (B) Heterogeneity in DNA repair inCHO cells exposed to 5 Gy and then allowed to repair for 10 and 30 min.The comets were classified in different classes according to tail area values.

levels of damage measured by the comet assay were similarto those detected by alkaline elution at pH 12.6 (Figure 5).This is not surprising since, although the pH of the cometassay is higher (13.5), the time of DNA exposure to alkali ismuch shorter than in the case of alkaline elution (40 minversus 16 h).

The repair kinetics were explored at two different doses ofENNG. As shown in Figure 6, after 4 h incubation of the cellsin fresh medium the majority of damage induced by a lowdose of ENNG (30 |iM) was repaired (Figure 6A), while 50%of the lesions induced by 60 (iM ENNG persisted in DNA(Figure 6B). In this case, the tail areas approached those ofthe untreated cells by 24 h after treatment. As in the case ofirradiated cells, the tail areas of repaired cells did not reach

ENU (mM)

B.3000

control 1mM 3mM 5mM

ENU dose

Fig. 3. ENU-induced DNA damage in CHO cells. (A) Survival curve. Eachpoint represents the mean of three independent experiments. SD values werealways <10%. (B) DNA breaks as measured by the comet assay. The tailareas of 50 comets per experimental point were measured by imageanalysis. The data are displayed as box plots (for details see the legend toFigure 1).

those of the untreated cells, even after 24 h repair time. Similarrepair kinetics were observed after cell exposure to ENU (datanot shown). These repair kinetics likely reflect removal ofN-ethyl-purines (mainly 3-ethylA and 7-ethylG) via baseexcision repair from DNA (Dogliotti et al., 1987; Vitelli et al.,1989). Since ENNG-induced DNA ssb are completely repairedwithin 24 h, it is unlikely that ethylphosphotriesters, whichpersist on DNA for long periods (Brent et al., 1988), contributesignificantly to DNA ssb detected by the comet assay.

It is well known that the rate of conversion of AP sites intochain breaks is accelerated at high pH (Lindahl and Andersson,1972). Accordingly, a higher level of DNA ssb is detectedafter alkylation damage by increasing the pH of the elutionbuffer in the alkaline elution assay or by lysing the cells inalkali for longer periods before sucrose gradient centrifugation(Figure 5; see also Dogliotti et al., 1984). In a previous studywe reported that DNA ssb detected in CHO cells after treatmentwith SN,-ethylating agents by alkaline elution at pH 12 werequickly resealed within 10 min after treatment. In contrast,DNA ssb measured at pH 12.6 were repaired at a much slowerrate (Dogliotti et al., 1984; Fortini et al., 1990). We decidedto explore the pH dependence of alkylation-induced DNA ssbas detected by the comet assay. CHO cells were treated with60 (iM ENNG and the level and repair of DNA ssb weremeasured by SCGE at pH 12.5. The data were compared withthose obtained by SCGE at pH 13.5 after cell exposure to thesame ENNG dose (compare Figure 6B and C). As shown in

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A. A.5000

control 30uM 30 mm 2hrs 4hrs 24hrs

B. B.

3000

2000

1000

0

I TT

T H ^

T j

l i--

5000

control 10uM 20uM 30uM 6OjiM

ENNGdose

Fig. 4. ENNG-induced DNA damage in CHO cells. (A) Survival curve.Each point represents the mean of three independent experiments. SD valueswere always <10%. (B) DNA breaks as measured by die comet assay. Thetail areas of 50 comets per experimental point were measured by imageanalysis. The data are displayed as box plots (for details see the legend toFigure 1).

control 60jiM 30 min 2hrs 4hrs 24hrs

c.

10

ASG(b)I

500

300 -

100

Fig. 5. ENNG-induced DNA ssb as measured by different alkaline methods.The number of ssb was measured by means of alkaline sucrose gradientcentrifugation after lysis in alkali for 21 (a) and 1 h (b) and by alkalineelution at pH 12.0 and 12.6 (data from Dogliotti et al., 1984).The dataobtained with the Comet assay were converted into ssb by calibration withthe dose-response curve obtained with X rays, by assuming that I Gyinduces 2.5 ssb per 1010 mol. wt of DNA. The lines drawn were obtainedby linear regression analysis.

Figure 6C, a lower level of DNA ssb was detected, and thesebreaks were completely resealed 2 h after treatment. The shapeof the comet tails was drastically affected by the pH of the

172

control 60uM 10min 1hr 2hrs

Fig. 6. Repair of ENNG-induced DNA damage in CHO cells. Cells weretreated with different concentrations of ENNG and then incubated at 37°C.At different time intervals samples were taken for SCGE. The comet assaywas performed under standard pH conditions, 13.5 (A and B), and atpH 12.5 (C). (A) After treatment with 30 \iM ENNG. (B and C) Aftertreatment with 60 |iM ENNG. The tail areas of 50 comets per experimentalpoint were measured by image analysis. The data are displayed as box plots(for details see the legend to Figure 1).

assay (Figure 7). The tail length was approximately twice thatmeasured at pH 13.5 while the tail area was one-tenth the size(Figure 7A and B). It might well be that under these pHconditions DNA strand separation is incomplete and only shortDNA molecules are free to expand and migrate.

These data indicate that lesions with different rates ofconversion into ssb at alkaline pH are repaired by the cells atdifferent rates and are therefore likely to belong to differentsubclasses of lesions.


DNA damage and repair by the comet assay

A. pH 12.53000

2000

1000

c o n t r o l 2mMENU

B. pH 13.5

Fig. 7. Microphotographs of CHO cells treated with 60 nM ENNG andsubjected to SCGE. (A) SCGE was performed at pH 12.5. (B) SCGE wasperformed at pH 13.5.

DNA damage profile by enzymatic detection of AP sitesAP sites are efficiently converted into ssb by enzymes calledAP endonucleases. We explored the feasibility of detecting therate of AP site formation after alkylation damage by inclusionwithin the cells of a bacterial AP lyase, endonuclease III. Thebreaks were then detected by the comet assay. This modifiedSCGE assay has been previously applied with success to thedetection of endogenous oxidative base damage in humanlymphocytes (Collins et at., 1993, 1995). Cells were treatedwith 2 mM ENU for 30 min at 37°C and, prior to SCGE, cellswere either incubated or not with endonuclease III. As shownin Figure 8, the incubation with the enzyme prior to SCGErevealed the presence of extra sites which were not convertedinto ssb by the exposure to the alkaline buffer of the cometassay alone. The presence of endonuclease Ill-sensitive siteswas also detected after 2 and 4 h of repair, clearly indicatingthe presence of persistent AP sites when the majority of breakswere already resealed. No endonuclease Ill-sensitive sites werepresent on DNA at 24 h post-treatment. These data indicatethat the repair of alkylation damage in CHO cells is completeby 24 h. Moreover, the lack of endonuclease Ill-sensitive sitesat this time confirms the specificity of the enzyme.

DiscussionBase excision repair is the major cellular defence against bothionizing radiation and alkylation damage. This repair pathwayinvolves an intermediate stage in which a DNA break ispresent. The rate of formation of DNA breaks is regulated bythe kinetics of the incision and resynthesis/ligation steps. Inparticular, the incision step is accomplished by the sequentialaction of specific DNA N-glycosylases, which recognize and

B.

I3 1000

! ! 15 5 S

5

Fig. 8. DNA breaks in CHO cells following exposure to 2 mM ENU for30 mm at 37°C. After treatment cells were incubated in fresh medium fordifferent periods of time. Before SCGE cells were incubated without or withendonuclease III. The tail areas of at least 50 comets per experimental pointwere measured by image analysis. (A) The data are displayed as box plots(for details see the legend to Figure 1). (B) The medians of the tail areavalues obtained in duplicate experiments are displayed.

100

<zo

6

I

I ' I I1 2 3 4

rapatrtbna (hrs)

Fig. 9. Repair kinetrcs of ENNG-induced DNA ssb as detected by differentalkaline methods. The DNA ssb were measured by SCGE at pH 13.5 and12.5, and by alkaline elution at pH 12.0 (data from Dogliotti et al., 1984).

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catalyze the removal of damaged bases, and of AP endo-nucleases, which cleave at the resulting base loss sites. Thegeneration of one or a few nucleotide-long gaps is promptlyfollowed by resynthesis of the damaged oligonucleotide byDNA polymerase(s) and ligation by DNA ligase to reconstitutethe original template (Dianov and Lindahl, 1994). The half-life in vivo of an abasic site is then the result of a dynamicprocess where breaks are formed and resealed at the same time.

Repair of X-ray-induced damage, as detected by SCGE,was rapid, with the majority of breaks resealed within 30 min.This is consistent with the rejoining rate of ssb.

The evaluation of alkylation damage and repair deservessome comment. Since alkylating agents do not induce ssbdirectly it is important to take into account that the level ofdetectable DNA lesions is dependent on (i) the repair rate; (ii)the half-life of AP sites and/or DNA breaks; and (iii) thealkali-lability of AP sites or of other alkyl-products. Since allssb detected by SCGE are repaired within 24 h after treatment,persistent lesions like alkylphosphotriesters are not convertedinto ssb under the alkaline conditions of this assay. Alkylationdamage detected by SCGE is confined to AP sites and breaksformed as intermediates in the base excision repair process.The rate of chemical hydrolysis of AP sites into ssb isdependent on pH. Accordingly, our data show that the pHconditions of the assay strongly affect the level of DNA breaksdetection. Interestingly, there is a correlation between alkali-lability of DNA lesions and their susceptibility to repair (Figure9). The lesions detected at pH 12.5 were completely repaired2 h after treatment, while the ssb detected at pH 13.5 werenot completely resealed even after 4 h. Moreover, in a previousstudy we have shown that ENU- and ENNG-induced ssb asdetected by alkaline elution at pH 12.0 are repaired with ahalf-time of 10 min (Dogliotti et al., 1984; Fortini et al.,1990). Taken together, these data suggest that different subsetsof lesions are detected under different • pH conditions. Wemight speculate that the localization of AP sites along thegenome affects both their rate of repair and their rate ofconversion into detectable ssb. It is not known whether thetail of the comet contains relaxed DNA loops attached to thenuclear scaffold (for a review see McKelvey-Martin et al.,1993) or DNA fragments with any contact with the nucleus.It is likely that it contains both. In fact, when breaks areintroduced into nucleoid DNA, a release of the supercoilingof the loop occurs, resulting in the extension of the loop towardsthe anode. If alkaline conditions are used, the unwinding willbe accelerated and strand separation will occur (Rydberg,1975). Therefore, both relaxed DNA loops and broken DNAfragments will be generated, thus increasing the amount ofDNA molecules able to migrate as comet tails. If repair eventsoccur preferentially in certain portions of the genome (for areview see Bohr et al., 1987) the formation of breaks will beclustered in certain sites. It is plausible that unwinding andstrand separation of DNA molecules close to the breaks, byrequiring rotation of short DNA molecules, would be favoured.Therefore, under milder alkaline conditions (pH 12-12.5), ssbat repair hotspots (breaks clustered in active genes?) would bepreferentially detected, while at pH 13.5 the number of ssbwould reflect mainly the repair rate of the genome overall.

An important conclusion of our study is that the level ofalkylation-induced damage as detected by SCGE is a measureof DNA repair events, not of actual damage. As in the caseof alkaline elution, the number of ssb detected corresponds to- 1 % of the alkylated products (Vitelli et al., 1989). This is

consistent with the repair rate of N-ethylpurines, which areremoved from CHO DNA with a half-life of 12 and 4 h for7-ethylG and 3-ethylA respectively (Dogliotti et al., 1987).

The level of ssb detected by SCGE was increased signific-antly by incubation of alkylated cells with endonuclease IIIprior to alkaline electrophoresis. These endonuclease Ill-sensitive sites should be AP sites which are not converted intossb by the exposure to alkali during SCGE. As expected, theirnumber decreases at increasing repair times and they disappear24 h after treatment, confirming that the repair process iscomplete. The inclusion within the cells of repair enzymes isan interesting tool for the construction of damage and repairprofiles of cells exposed to a variety of physical and chemicalagents (Collins et al., 1993, 1995; Epe et al., 1993a, b).

Finally, caution should be taken when SCGE is used tomonitor interindividual differences in DNA damage. As in thecase of alkylation damage, it might well be that the numberof ssb detected is not a measure of actual DNA damage butis a narrow window opened on the complex dynamics of thecellular repair processes.

AcknowledgementsWe thank L.Gargano for technical assistance and M.Bignami for criticallyreading the manuscript. This work was partially supported by ECC Contractno EV5V-CT92-O223.

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Received on August I, 1995; accepted on October 26, 1995

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analysis of dna alkylation damage and repair in mammalian cells

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