N-Ethyl-N-nitrosourea predominantly induces mutations at AT base pairs in pre-meiotic germ cells of Drosophila males

Molecular mutation spectra induced by N-ethyl-N-nitrosoureahave been obtained in several organisms and test systems, frequentlyshowing different results.In Drosophila melanogaster this spectrumhas been analyzed in postmeiotic stages, resulting in good agreementbetween the adduct spectrum and mutational events, the majoritybeing GCAT transitions (61%).However, when collecting data aboutin vivo ENU-induced mutations in mouse germ cell stages mostlydamage at A:T sites (89%) was observed. In this work we analyzethe molecular spectrum induced with ENU in pre-meiotic repair–activemale germ cells of D.melanogaster, using the specific locustest (SLT) with the vermilion locus as target. Results showthat the most mutagenic sites in spermatogonial stem cells ofDrosophila are A:T pairs (85%), with ATTA transversions (50%)and ATGC transitions (35%) as the most frequent mutations.Differencesfrom the post-meiotic spectrum may be explained by the activerepair of some adducts, such as O⁶-ethylguanine and N-alkyl-inducedabasic sites. In addition, these results show the relevanceof the minor lesions O⁴-ethylthymine and O²-ethylthymine inthe production of mutations, as a consequence of their poorrepair.Finally, since there is a striking similarity to theENUinduced mutation spectrum in mouse, these results revealthat Drosophila continues to be an excellent model system. ¹To whom correspondence should be addressed. Tel: +34 8 510 3599; Fax: +34 8 510 3534; Email:msierra{at}sauron.quimica.uniovi.es     

Effect of nucleotide excision repair on hprt gene mutations in rodent cells exposed to DNA ethylating agents

The role of the nucleotide excision repair (NER) pathway inremoval of DNA ethylation damage was investigated by means ofhprt mutational spectra analysis in the NER-deficient Chinesehamster ovary cell line UV5, which lacks ERCC2/XPD, and in itsparental cell line AA8. Both cell lines were exposed to ethylmethanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU). EMS gavea similar dose-dependent increase in hprt mutants in UV5 comparedwith AA8. In both cell lines EMS-induced mutations in the hprtcoding region consisted almost exclusively of GC     

DNA sequence analysis of in vivo hprt mutation in human T lymphocytes

We have determined the molecular basis of hypoxanthine-guaninephosphoribosyltransferase (hprt) mutations that arose in vivoin the T lymphocytes of a normal male subject. In previous studies16% (23/141) of the mutants from this individual analyzed bySouthern blot displayed large structural alterations in hprt.Thirty-two mutants without these large hprt structural alterationsproduced sufficient hprt cDNA for polymerase chain reactionamplification and DNA sequence analysis. Base substitutionsin hprt cDNA resulting in missense mutations and one mRNA splicingaberration (inclusion of intron sequences) were observed in18/32 of these these mutants; substitutions occurred at bothAT and GC base pairs. Small deletions (3/32), a tandem changeand a single base insertion were also observed among the hprtcDNAs. Exon skipping and inclusion of hprt intron sequencesin the hprt cDNA were observed in an additional 9/32 of themutants. Analysis of T cell receptor (TCR) gene rearrangementsrevealed that six of eight mutants with an identical hprt T—Atransversion displayed the same TCR rearrangement pattern, indicatingthat they were clonally related and arose from a single in vivomutational event. ³Present address: Department of Pathology, University of NorthCarolina Chapel Hill, NC 27514, USA ⁴To whom correspondence should be addressed      

Molecular characterization of mutations in the hprt gene of normal human skin keratinocytes treated with N-ethyl-N-nitrosourea: Influence of O6-alkylguanine alkyltransferase

O⁶-Alkylguanine-DNA alkyltransferase (AGT) is responsible for repairing the O⁶-alkylguanine lesion in DNA. There is wide variation in the levels of AGT between organ and cell types, which appears to correlate with cell and tissue type sensitivity to the mutagenic and carcinogenic effects of alkylating agents. In order to investigate the role of AGT in modulating the frequency and types of mutations induced in one type of normal human parenchymal cells, we examined the types and frequency of mutations in the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene in 116 mutants derived from two N-ethyl-N-nitrosourea (ENU)-treated normal human skin keratinocyte cell lines. O⁶-Benzylguanine (O⁶-BZ; 5 μM × 2 hours) was used to specifically inhibit AGT activity before ENU treatment (0 to 5 mM × 1 hour). O⁶-BZ increased both the cytotoxic and mutagenic effects of ENU by 1.8- and 3- to 5-fold, respectively. In both treatment groups, most of the mutations were base substitutions (72%). The proportion of GC to AT transitions in the O⁶-BZgroup (14/31) was twice that in the group treated with ENU alone, consistent with the loss of AGT activity in these cells. There was no strand specificity of GC to AT and AT to GC transitions in both groups. Base transversions accounted for 28% of total base substitutions. A lower than expected proportion of AT to TA transversions were observed in both cell lines, which decreased in the O⁶-BZ pretreated group. A strand bias was observed for GC to TA and AT to TA transversions. Most of the G to A and G to T base substitutions had one or more purines flanking 3′ to the mutated deoxyguanosines. There were more deletion mutants with the deletion of exon 1, 4, 6, and 8 in the BZ group than in the control group. These data, characterizing the mutational spectra of ENU in normal human keratinocytes treated in vitro, indicate that GC to AT and AT to GC transition mutations predominate in these cells depleted or not depleted of AGT. Environ. Mol. Mutagen. 29:168–179, 1997. © 1997 Wiley-Liss, Inc.     

Spontaneous mutation spectrum in the hprt gene in human lymphoblastoid TK6 cells

A spectrum of 100 mutations in the endogenous hprt gene of thehuman lymphoblastoid TK6 cell line is presented. The majorityof the mutations originates in sequences outside the codingregion of the gene. Large deletions are a major cause of inactivationof the hprt gene (57% of the mutants). Mutations in the splicesites that result in several forms of aberrantly spliced mRNAare relatively frequently recovered (16%) compared with mutantscontaining alterations in the coding region of the hprt gene(27%). The majority, but not all, of the splice mutants containan alteration in the consensus sequences of the splice sites.A spectrum of mutations in the coding region of the hprt geneenlarged to a total of 42 mutants shows that basepair substitutionspredominate (71%) and that small deletions and insertions areless frequently recovered. Basepair substitutions arise slightlymore frequently at GC basepairs than at AT basepairs. ³To whom correspondence should be addressed     

Chloroacetaldehyde-induced mutagenesis in Escherichia coli: specificity of mutations and modulation by induction of the adaptive response to alkylating agents

The mutational specificity of chloroacetaldehyde (CAA), oneof the metabolites of the human carcinogen vinyl chloride (VC),has been determined through the examination of Arg⁺ revertantsin Escherichia coli AB2497 (Arg^–) and identification oftheir tRNA suppressors. The predominant mutations were GC ATtransitions (65%) followed by AT TA transversions (12.5%).The observed mutational specificity of CAA is very similar tothe reported specificity of the other VC metabolite, chloroethyleneoxide. The induction of the adaptive response to alkylatingagents significantly decreased the frequency of CAA-inducedRif^r and Arg⁺ mutants in E.coli AB2497 and increased the cellsurvival. Likewise, the adaptation of bacterial cells decreasedthe frequency of GC AT transitions in CAA-treated M13glyU phagetransformed to E.coli JC15419 and increased the phage survival.Experiments with strain MS23, which is an alkA mutant deficientin 3-methyladenine-DNA glycosylase II, and with MS23 harboringthe pYN1000 plasmid carrying the alkA⁺ gene, have shown thatinduction of this repair enzyme is responsible for reductionof the level of CAA-induced mutations. The role of N², 3-ethenoguanine,among the other etheno-adducts, in CAA-induced mutagenesis andas a target for repair in 3-methyladenine-DNA glycosylase IIproficient bacterial cells is discussed. ¹To whom correspondence should be addressed     

Analysis of 4-nitroquinoline-1-oxide induced mutations at the hprt locus in mammalian cells: possible involvement of preferential DNA repair

Mutation spectra induced by 4-nitroquinoline 1-oxide (4NQO)at the hprt locus for both normal (AA8) and 4NQO-sensitive (UV5)Chinese hamster ovary cells were determined to investigate theeffect of DNA repair on the nature of induced mutations. TheUV5 cell line is three times more sensitive to 4NQO than theAA8 parental cell line. In UV5 cells, the dGuo-N2-AQO adduct,which is considered to be the most toxic and mutagenic adductin Escherichia coli, is poorly repaired. The molecular natureof 30hprt mutants isolated from AA8 and 20 isolated from UV5cells was determined by sequence analysis of in vitro amplifiedhprtcDNA. Both similarities and differences emerged. In bothcell lines we found that (i) 4NQO is basically a base substitutionmutagen acting almost exclusively at G residues and (ii) G transversionsare prevalent over G transitions in both cell lines, independentlyfrom the ability to repair dGuo-N2-AQO. A high proportion (13/25)of splice mutations was observed in AA8 cells, statisticallydifferent (P < 0.04, Fisher‘s exact test) from theincidence of splice mutants in UV5 cells (4/20). In AA8 mutants,all but two of the point mutations were due to lesions localizedon the non-transcribed strand, suggesting preferential repairof the transcribed strand. Compared with AA8, the proportionof mutants due to lesions present on the transcribed strandwas higher in UV5 cells, as expected if a preferential repairmechanism was impaired in the sensitive cell line. Our dataare consistent with the molecular defect in DNA repair recentlycharacterized in UV5. ³To whom correspondence should be addressed     

Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations

To study in vivo somatic mutations a C57BL/6 transgenic mousemodel was constructed harboring multiple chromosomally integratedcopies of the plasmid pUR288, which carried the lacZ reportergene as the mutational target. We previously demonstrated thatlacZ-containing plasmids could be rescued from their integratedstate efficient enough to detect mutations in lacZ by positiveselection. The smaller size of the plasmid vector, as comparedwith our earlier transgenic mouse model based on bacteriophagelambda vectors, should offer considerable advantages in termsof rescue efficiency and sensitivity to large size alterationsin the lacZ gene. To evaluate the plasmid-based mouse modelfor its suitability to detect in vivo mutations, we determinedmutant frequencies in different organs of untreated and ethylnitrosourea (ENU)-treated animals using a new, improved protocol.The rescue efficiencies obtained were as high as 200 000/µggenomic DNA; millions of transformants could be obtained inone single experiment. The average spontaneous mutant frequencyin four different organs of 4- to 8-week-old mice ranged from4.41 to 6.82x10^–5;, compared with a mutant frequency ofthe same plasmid grown in Escherichia coli of     

Strand-specific mutation induction by 1,2-dibromomethane at the hypoxanthine-guanine phosphoribosyltransferase locus of Chinese hamster ovary cells

The nature of mutations induced by 1,2-dibromoethane (DBE) atthe hprt (hypoxanthine-guanine phosphoribosyltransferase) genewas analysed in Chinese hamster ovary (CHO-9) cells. Molecularcharacterization of 36 hprt mutants at the cDNA level yielded19 GCAT transitions, two ATCG transversions, three frameshiftmutations, two identical small deletions and 10 exon deletions.Further analysis of the deletion mutants by amplification ofspecific exons from genomic DNA showed two more GCAT transitionsat splice sites and an {small tilde}70 bp deletion. Assumingthat the S-[2-(N7-guanyl)ethyl]glutathione adduct is responsiblefor the GCAT transitions, 90% of the affected guanines werelocated in the non-transcribed strand of the hprt gene, suggestinga strand bias in repair of this adduct Nearest neighbour analysisof induced GCAT transitions indicates a preference for a 5'-PyPuGDNA sequence, i.e. 15/21 mutated guanines were located in eithera TGG or a CAG DNA sequence. These molecular data on DBEinducedmutations showed similar features as data from a study by Graveset al (Mutagenesis, 11, 229–233, 1996) in which they analyzed13 hprt mutants induced by DBE in CHO-K1 cells. Six of the sevenGCAT mutations were on positions mutated more than once amongthe 36 hprt mutants in the present study. The combined findingssuggest that some positions seem to be hot spots for DBE-inducedmutations. Concerning the relevance of these in vitro studiesfor germ cell mutagenesis the conclusion may be that these datalend further support to the view that mutation spectra derivedfrom in vitro systems have little predictive value for the natureof mutations induced in post–meiotic germ cells in vivo,as demonstrated for other alkylating agents in both Drosophitaand mice. ¹To whom correspondence should be addressed. Tel: +31 071 5276145; Fax: +31 071 5221615; Email: nivard{at}rullf2.medfac.leidenuniv.nl     

Mutation induction by UV light in retroviral hprt cDNA integrated at various chromosomal positions in repair-deficient hamster cells

Mutation induction by UV irradiation was studied in a retroviralvector integrated in one copy per cell at various chromosomalpositions. As a mutational target, hamster hprt cDNA was presenton the retroviral vector. To minimize the influence of repairwe used repair-deficient hamster cells, V-H1 and UV5, as a recipientfor the vector. There is no major influence of chromosomal positionon UV-induced mutation frequency and spectrum because no statisticallysignificant difference between mutation induction in retroviralcDNA copies integrated at different chromosomal sites was observed.However, a major difference was found in mutation inductionbetween the endogenous hamster hprt gene and the retroviralcDNA copies. Most noticeable was the absence in the cDNA ofthe strong strand bias for mutation induction, which was reportedfor the endogenous hprt gene. Our results with the hprt cDNAexclude as a general phenomenon a difference in mutation inductionfor leading and lagging strand DNA replication, which was proposedas an explanation for this strand bias in the endogenous gene.The similarity of mutation induction in the different retroviralcDNA copies, all directly surrounded by the same DNA sequenceelements, together with the marked difference between the mutationinduction in the endogenous gene and the cDNA copies may pointto an important role of chromatin structure in mutation induction.     

Molecular analyses of in vivo hprt mutations in human T-lymphocytes I. Studies of low frequency 'spontaneous' mutants by Southern blots

Fifty wild-type and 164 in vivo-derived hprt mutant T-cell clonesobtained from eight non-mutagen-exposed adult males with mutantfrequency values in the normal range (usually < 10 x 10^–6)were studied by Southern blot analyses to determine frequencyand extent of gross structural alterations in the hprt gene.Sixteen (9.8%) of the mutant clones showed hprt changes. Nosite or type of lesion predominated. Relative frequencies ofgross structural alterations in the recovered hprt mutants didnot differ among the eight individuals, within limits detectableby the study. DNA from 201 of these 214 clones was also studiedwith a T-cell receptor (TCR) ß gene probe as a markerfor independence of in vivo-derived clones. Some clones werealso studied with a TCR gene probe. Ninety-four percent ofwild-type and 89% of the hprt mutants were found to originatefrom independent in vivo precursors. Therefore, most of therecovered hprt mutants in the study were presumably derivedfrom separate in vivo mutations. For non-mutagenized adultswith normal mutant frequencies, in vivo mutant frequencies arethus reasonable approximations of in vivo mutation frequencies,although elsewhere we show that this is not necessarily truefor individuals with grossly elevated mutant frequencies.     

DNA adducts,mutant frequencies,and mutation spectra in various organs of λlacZ mice exposed to ethylating agents

To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, λlacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O⁶-ethylguanine (O⁶-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O⁶-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O⁶-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC → AT transitions, consistent with the O⁶-EtG lesion, and 28% TA → AT transversions, expected from O²-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC → AT mutations and 22% were TA → AT mutations. This suggests that in bone marrow O⁶-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O⁶-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O⁶- and N7-adducts were present. Environ. Mol. Mutagen. 31:18–31, 1998. © 1998 Wiley-Liss, Inc.     

The molecular nature of mutations induced by adriamycin at the hprt locus of V79 cells

Adriamycin (AM), a widely used chemotherapeutic drug, induceda broad spectrum of gene mutations at the hprt locus of V79cells. The frequency and distribution of AM-induced deletionswas analyzed with multiplex polymerase chain reaction in twoV79 cell lines, which differed considerably in their spontaneousdeletion frequency. Among AM-induced mutants, deletions predominatedin both cell lines. Apart from total deletions of the hprt gene,partial deletions were found which were distributed all overthe hprt gene with breakpoints in nearly all introns. Underthe same experimental conditions, chromosome aberrations wereinduced by AM which mainly represented chromatid-type aberrations.Neither the induction of gene mutations nor the induction ofchromosome aberrations was enhanced by the repair inhibitor3-aminobenzamide. These results are discussed in the contextwith our earlier findings on bleomycin-induced mutations andit is suggested that at least two mechanisms lead to the formationof gene deletions. One of them seems to be associated with amisrepair process of frank DNA double-strand breaks and relatedto chromosome aberrations while the other is not. ¹To whom correspondence should be addressed     

Quantitative relationship between ethylated DNA bases and gene mutation at two loci in CHO cells

In a previous study we showed that the formation of O⁶-ethylguanine (O⁶-EtGua) in the DNA of CHO cells in culture correlated with mutations induced by ethylnitrosourea (ENU) and diethylsulfate (DES) at the hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus but not at the Na, K-ATPase locus. This study was extended to another ethylating agent, ethyl methanesulfonate (EMS). DNA adduct formation and induction of mutation at the two gene loci were determined simultaneously in CHO cells after EMS exposure. The extent of ethylation at the N⁷ and O⁶ positions of guanine and at the N³ site of adenine were measured and the possible correlations with 6-thioguanine resistance (6-TG^r) and ouabain resistance (oua^r) mutations were investigated. A good correlation between the levels of ethylation at O⁶ guanine and mutation frequency at hprt gene by all three ethylating agents was observed. In the case of the oua^r locus, the frequency of O⁶-EtGua adducts correlated with mutation induction by EMS and ENU but not by DES. Although both EMS and DES have similar reaction mechanisms, these results highlight differences in their mutational specificity. The comparison of this type of analysis with mutational spectra revealed that correlation studies may be inadequate to analyse multi-component phenomena like mutation formation. © 1993 Wiley-Liss, Inc.     

Concentrated drinking water extracts, which cause bacterial mutation and chromosome damage in CHO cells, do not induce sex-linked recessive lethal mutations in Drosophila

Concentrated extracts prepared from chlorinated drinking watersamples were tested for their ability to induce sex-linked recessivelethal mutations in Drosophila melanogaster. Adult flies wereallowed to feed on sucrose solutions prepared using neat orhalf-strength water extract. The drinking water extracts usedfor this study were also tested in bacterial fluctuation assaysusing Salmonella typhimurium TA100 and TA98 and in an in vitrocytogenetic assay using CHO cells. Although the water extractsgave positive results in both of these in vitro tests, therewas no evidence of mutagenic activity in the Drosophila studies. ¹Present address: Glaxo Group Research Ltd, Park Road, Ware,Hertfordshire SG12 ODJ, UK      

Spectrum of spontaneously occurring mutations in the HPRT gene of the Chinese hamster V79 cell mutant V-H4, which is homologous to Fanconi anemia group A

The mitomycin C (MMC)-hypersensitive Chinese hamster V79 cellmutant V-H4 has a cellular phenotype similar to Fanconi anemia(FA), and has been shown to be homologous to FA group A. Toexamine consequences of the defect in V-H4 cells on spontaneousmutagenesis, we studied the frequency and nature of spontaneousmutations at the hypoxanthine phosphoribosyltransferase (HPRT)locus in this mutant and the parental V79 cells. The mutationrates expressed as the number of mutations per cell per generationwere 8.7 X10^–7 and 3.7 X10^–7 for V-H4 and V79 cellsrespectively. The molecular spectrum of 42 spontaneous hprtmutants of V-H4 cells was determined and compared with the previouslydescribed spectrum of spontaneous mutations at the HPRT locusof Chinese hamster V79 cells. The spectra of spontaneous mutationsin the hprt gene of both cell lines are predominated by basepair substitutions and splice mutations. Among the base changes,V-H4 shows a larger frequency of transitions (13/42; 31%) thantransversions (3/42; 7%), whereas in V79 transversions are observedmore often than transitions (P < 0.001; Wilcoxon test). Thefrequency of splice mutations in V-H4 (17/42; 40%), which affectsexon 4 almost exclusively, is not significantly different fromV79. The fraction of deletions in V-H4 is low (6/42; 14%), andcomparable to the level in V79. This is in contrast with thepublished molecular spectrum of spontaneous hprt mutants inFA (group D) cells, which consists predominantly of deletions. ⁴To whom correspondence should be addressed at MGC-Department of Radiation Genetics and Chemical Mutagenesis     

DNA sequence analysis of spontaneous mutations at a LacZ transgene integrated on the mouse X chromosome

Transgenic mice with integrated shuttle vectors containing theLacZ mutational target gene were used to study spontaneous mutationalevents in vivo. The transgenic mouse strain used carries theLacZ transgene on the X chromosome and was previously foundto be characterized by 25–fold higher spontaneous mutationfrequency in liver and brain compared with at least three othertransgenic mouse strains. To determine the nature of in vivospontaneous mutational events, 35 mutant LacZ genes isolatedfrom liver and brain of mice from strain 35.5 were analyzedat the DNA sequence level. The results obtained indicate thatsingle base-pair changes were predominant in both liver andbrain. However, in liver the majority of mutations were transitionswhereas in brain transversions were predominantly observed.Six mutants appeared to contain multiple dispersed mutations,separated by as much as 44 bp. Mutations were generally locatedwithin a 500 bp region encoding the active site of the ß-galactosidaseprotein. Our results indicate that spontaneous mutations atthe LacZ transgene are tissue specific and dependent on thechromosomal position of the LacZ transgene. ³To whom correspondence should be addressed at: Harvard Medical School, Beth Israel Hospital, 330 Brooldine Avenue, Boston, MA 02215, USA     

An assay for loss of heterozygosity in vivo at the Dlb-1 locus

Loss of heterozygosity (LOH) is a frequent event in many tumours,resulting in the loss of tumour suppressor genes and ultimatelymagnifying the number of cells with a mutant phenotype. We haveused the Dlb-1 locus as a simple quantitative assay for LOHin vivo. Mutations of the dominant Dlb-1^b allele are readilydetected in heterozygous (Dlb-1^a/Dlb-1^b) mice by the loss ofhistochemical staining, which results in unstained, white (Dlb-1^a/Dlb-1^–)ribbons on a stained background. Such ribbons are extremelyrare in untreated C57BL mice which are homozygous for the dominantallele, as would be expected when two independent mutationsare required. To test for LOH, we first treated the animalswith a high dose of ethylnitrosourea (ENU) which induces manymutations and thus many heterozygous cells, and allowed 2 weeksfor gene expression. Then the animals were treated with thetest agent to determine if it could cause LOH and thus convertheterozygous mutant cells, which would not produce detectableribbons, into homozygotes that would produce white, non-stainingribbons. Treatment with ENU alone produced a low, but detectable,frequency of mutant ribbons. Treatment with X-rays alone producedno detectable increase in the frequency of mutant ribbons. Combinationsof these treatments produced additive effects, thus showingthat no significant LOH was induced. The additivity of the twoequal ENU treatments was unexpected, since double mutants shouldincrease as the square of the mutant frequency. This can beexplained in two ways: (i) stem cells are immutable except whencycling, which is rare, so few stem cells are hit twice or (ii)there is a constant rate of LOH which is not significantly increasedby either X-rays or ENU. The assay can be used to determineif other factors can induce significant frequencies of LOH. ¹To whom correspondence should be addressed     

Elevated intracellular dCTP levels reduce the induction of GC -> AT transitions in yeast by ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine but increase alkylation-induced GC -> CG transversions

The effect of an increased intracellular dCTP:dTTP ratio onthe specificities of ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) mutagenesis was examined in the yeast Saccharomyces cerevisiae.To do so, we used a dCMP deaminase-deficient (ded1) strain havinga dCTP:dTTP ratio >77-fold larger than its isogenic wild-typeparent under the treatment conditions employed. This DNA precursorimbalance lowered the frequencies of EMS- or MNNG-induced SUP4-omutations by 75 or 45% respectively, relative to the correspondingvalues for the wild-type strain. A total of 405 SUP4-o mutationsproduced by the alkylating agents in the dcd1 background werecharacterized by DNA sequencing and the mutational spectra werecompared to those for 399 mutations induced in the wild-typeparent and 207 mutations that arose spontaneously in the dcd1strain. Unexpectedly, the frequencies of EMS- and MNNG-inducedGC AT transitions in the dcd1 strain were found to be reducedby 93 and 68%, respectively, considerably more than the decreasesfor the overall SUP4-o mutation frequencies. The differenceswere due mainly to substantial increases in the frequenciesof GC CG transversions. Although these events were the predominanttype of spontaneous substitution in the dcd1 strain, they weremore frequent after alkylation treatment and were distributeddifferently than the spontaneous GC CG transversions. Preferencesfor the EMS- or MNNG-induced GC AT transitions to occur atGC sites having the guanine located on the transcribed strandor preceded by a 5' purine, respectively, also were diminishedin the dcd1 strain. Together, these findings support mispairingof O⁶-alkylguanine with thymine as the cause of most EMS andMNNG mutagenesis in wild-type yeast. However, the data alsolead us to suggest that the normally error-free repair of apurinicsites resulting from the lability or removal of EMS- or MNNG-inducedN⁷-alkylguanine can be rendered errorprone by increasing thedCTP pool. ¹Present address: Department of Biology, The University of Saskatchewan,Saskatoon, Saskatchewan S7N 0W0, Canada ²To whom correspondence should be addressed