Vol. 172, No. 6JOURNAL OF BACTERIOLOGY, June 1990, p. 3009-30140021-9193/90/063009-06$02.00/0Copyright © 1990, American Society for Microbiology

Specificity of the Mutator Effect Caused by Disruption of the RADIExcision Repair Gene of Saccharomyces cerevisiae

BERNARD A. KUNZ,* LESTER KOHALMI, XIAOLIN KANG, AND KATHRYN A. MAGNUSSONDepartment of Microbiology, The University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2

Received 11 December 1989/Accepted 20 March 1990

- Disruption of RADI, a gene controlling excision repair in the yeast Saccharomyces cerevisiae, increased thefrequency of spontaneous forward mutation in a plasmid-borne copy of the SUP4-o gene. To characterize thiseffect in detail, a collection of 249 SUP4-o mutations arising spontaneously in the radl strain was analyzed byDNA sequencing. The resulting mutational spectrum was compared with that derived from an examination of322 spontaneous SUP4-o mutations selected in an isogenic wild-type (RADI) strain. This comparison revealedthat the radl mutator phenotype was associated with increases in the frequencies of single-base-pairsubstitution, single-base-pair deletion, -and insertion of the yeast retrotransposon Ty. In the radl strain, therelative fractions of these events and their distributions within SUP4-o exhibited features similar to those forspontaneous mutagenesis in the isogenic.RADI background. The increase in the frequency of Ty insertionargues that Ty transposition can be activated by unrepaired spontaneous DNA damage, which normally wouldbe removed by excision repair. We discuss the possibilities that either translesion synthesis, a reduced fidelityof DNA replication, or a deficiency in mismatch correction might be responsible for the majority ofsingle-base-pair events in the radl strain.

The product of the RADI gene of the yeast Saccharomy-ces cerevisiae is believed to function at the incision step ofexcision repair ofDNA damage (9). Thus, it is not surprisingthat defects in this gene increase sensitivity to a variety ofDNA-damaging agents, including UV, the UV-mimeticchemical 4-nitroquinoline-1-oxide, mono- and bifunctionalalkylating agents,.and photoactivated psoralens (6, 8, 9, 40,41, 49). As well, the RADI gene product is required for therepair of NM-methyladenine (13). Not only do RADI deficien-cies sensitize cells to the lethal effects of certain genotoxicagents, but also they enhance UV and 4-nitroquinoline-1-oxide-induced mutagenesis (27, 40) and UV-induced mi-totic interchromosomal recombination (45). Recent findingssuggest that the RADI gene product also plays a role inmitotic intrachromosomal recombination and in integrationof linear DNA molecules into homologous genomic se-quences (2, 17, 44). In addition to these various properties,defects in RADl confer a mutator phenotype (35, 43, 47).Both enhanced locus reversion to prototrophy and forwardmutation to suppression and canavanine resistance havebeen reported, but neither the precise mutational changesinvolved nor the specificity of the mutator effect has beenelucidated.To account for the association between repair defects and

enhanced spontaneous mutagenesis in yeast cells, von Bor-stel and colleagues (12, 42) have invoked the repair channel-ing hypothesis (5, 10). According to this hypothesis, impair-ment of a specific repair pathway results in channeling ofdamage normally repaired by that pathway along other,competitive pathways. In a similar fashion, shunting ofspontaneous DNA lesions through mutagenic repair path-ways might account for elevated spontaneous tnutation inrepair-deficient strains. However, the nature of these error-prone pathways has remained obscure, and generally themagnitudes of the mutator effects in yeast cells are relativelysmall. Consequently, it has been suggested that repair de-fects might have a more indirect influence on spontaneous

* Corresponding author.

mutation, perhaps by somehow reducing the fidelity. ofDNAreplication (43) or the efficiency of correcting replicationerrors (26). Alternatively, the products of some repair genesmight play a regulatory role (7, 16) so that modulation ofprocesses other than DNA repair could be responsible forthe mutator phenotypes of certain repair-deficient mutants.

Characterization of the locations and types of DNA se-quence alteration occurring spontaneously within a singlegene in a radl background would provide valuable infoi-ma-tion about the specificity of the radl mutator effect. In turn,this could yield important clues about the mechanism(s) ofenhanced spontaneous mutagenesis in excision repair-defi-cient strains. To examine mutational specificity, we previ-ously developed a system for the DNA sequence analysis offorward mutations in the yeast tRNA suppressor geneSUP4-o (39). In this system, all types of base pair substitu-tion as well as deletions, duplications, insertions, and morecomplex events can be identified (11, 20, 22). Here, wereport our use of this system to characterize spontaneousSUP4-o mutations arising in a radl mutator strain. Compar-ison of the spectrum of mutations obtained in this strain withthe corresponding spectrum for an isogenic wild-type strainrevealed that the radl mutator phenotype was associatedwith enhanced frequencies of single-base-pair substitution,single-base-pair deletion, and insertion of the yeast retro-transposable element Ty. We consider the possible mecha-nisms that might give rise to the majority of single-base-pairevents in the radl strain and suggest that spontaneous DNAdamage, which is a substrate for excision repair, can activateTy transposition.

MATERIALS AND METHODSStrains, plasmid, and media. The haploid, repair-proficient

yeast strain MKP-o (MATa canl-100 ade2-1 lys2-1 ura3-52leu2-3,112, his3-A200, trpl-A901) has been described (39).KAM1, carrying a radl ::LEU2 insertion but otherwise iso-genic to MKP-o, was derived by transforming MKP-o with a6.96-kilobase (kb) Sall DNA fragment encompassing theRADI gene and flanking regions and having the internal

3009

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

3010 KUNZ ET AL.

2.1-kb StuI-Clal fragment ofRADl (70%o of the RADI codingsequence) replaced with a 3.2-kb BglII fragment containingthe LEU2 gene. The 6.96-kb Sall fragment was obtainedfrom plasmid pL962 (provided by R. L. Keil). MKP-o wastransformed by using lithium acetate (15), and colonies thatemerged on leucine omission medium were tested for sensi-tivity to 254-nm UV. Replacement of the RAD1 gene with asingle copy of the 6.96-kb Sail fragment in the highly UVsensitive transformant KAM1 was confirmed by DNA hy-bridization analysis (data not shown). Plasmid YCpMP2 wasthen transformed into MKP-o and KAM1 to make strainsMKP-op and KAM1-p, respectively. Escherichia coliJF1754 (Alac gal metB leuB hisB436 hsdR) was used forplasmid recovery from yeast cells. Plasmid YCpMP2 (39) isa centromere-containing vector having components thatenable it to replicate autonomously in yeast and bacterialcells. In yeast cells, such plasmids exhibit typical chromatinorganization, replicate once per cell cycle in S phase, and aremaintained predominantly as single copies in haploid cells(37). YCpMP2 carries SUP4-o, an ochre suppressor allele ofa yeast tyrosine tRNA gene. It also bears the yeast URA3,gene which permits selection for YCpMP2 via complemen-tation of the ura3-52 allele present in MKP-o and KAML.Media for growth of yeast and bacterial cells and for mutantselection and characterization have been described (21, 39).

Detection of SUP4-o mutants. Selection of forward muta-tions in the SUP4-o gene is based on loss of suppressoractivity. MKP-op carries ochre-suppressible markers thatconfer canavanine resistance (canl-100), red pigmentation(ade2-1), or lysine auxotrophy (Iys2-1). Normally, thesemarkers are suppressed by SUP4-o so that cells carryingYCpMP2 are sensitive to canavanine and form white, lysine-independent colonies. Reduction of suppressor activity leadsto the formation of red, canavanine-resistant colonies thatrequire lysine. This procedure can detect at least a 30%decrease in the production of functional suppressor tRNA(48). Scoring for the loss of suppression of all three markersis unlikely to bias the recovery of mutations significantly.Mutations that prevent the suppression of just two ochremutations are rare (1% of mutations analyzed) at the chro-mosomal SUP4-o locus (24), and we have not detected DNAsequence alterations in the plasmid copy of the SUP4-o genefrom mutants isolated only on the basis of canavanineresistance and red coloring (23). Presumably, such mutantsare due to antisuppressors (30). Furthermore, we haverecovered mutations at all but 6 of the 75 exon sites in thegene and have identified a wide range of mutational classesby the selection protocol used here (20; B. A. Kunz,unpublished data). In particular, we have detected single-base-pair changes at 40 of the 41 G *C pairs (G- C A T

transitions, 34 sites; G*C -* C G and G *C T*Atransversions, 38 sites each) and at 29 of the 34 A T pairs(A T- G C transitions,23 sites;A*T-C GandA T

-*T A transversions, 16 and 20 sites, respectively) withinthe SUP4-o exons. We have also found the three possiblesubstitutions at position 51 (G - C pair) within the 14-base-pair intron of SUP4-o. In addition, an A TT -- T- A

transversion has been detected at position 43 of the chromo-somal SUP4-o gene (25). Consequently, although SUP4-o isjust 89 base pairs long, at least 172 substitutions at aminimum of 71 sites lead to detectable phenotypic changes.These findings argue against selection bias.

Plasmid stability and spontaneous mutation. Cultures ofMKP-op or KAMl-p were grown from low-titer inocula (33cells per ml) in uracil or uracil-leucine omission broth,respectively, at 30°C with shaking to final titers of approxi-

TABLE 1. Characterization of canavanine-resistant colonies

Strain Mutant No. examined' FrequencybStrain typesa (% of total) (10-6)MKP-op (RADI) Total 29,325 (100) 72

White 26,569 (90.6) 65Red 2,756 (9.4) 6.7Red, Lys- 836 (2.8) 2.01

KAMl-p (radl) Total 37,316 (100) 360White 35,761 (96) 346Red 1,555 (4.2) 15Red, Lys- 1,358 (3.6) 13.1

a The total includes canavanine-resistant mutants that are (i) white or (ii)red and Lys' or Lys-.

I Colonies were isolated from, and frequencies are the means for, 40(MKP-op) or 22 (KAMl-p) independent cultures.

mately 107 cells per ml (determined by Coulter Counter).Then the cell suspensions were diluted when necessary andplated on uracil (MKP-op) or uracil-leucine (KAM1-p) omis-sion medium to measure viability, on minimal mediumsupplemented with uracil and other nutrients to determineplasmid retention, and on uracil or uracil-leucine omissionmedium containing canavanine (30 ,ug/ml) to select canava-nine-resistant mutants. All plates were incubated for 6 daysat 30°C. Plasmid stability was expressed as the ratio [(num-ber of colonies on minimal omission medium)/(number ofcolonies on minimal medium containing uracil)] x 100. Red,canavanine-resistant colonies were subcultured to uracilomission medium, incubated for 2 days at 30°C, replicated touracil-lysine omission medium, and incubated at 30°C for 2to 3 days. Lysine auxotrophs were scored as SUP4-o mu-tants. The mutation frequencies were then determined foreach independent culture, and the rate of mutation pergeneration was calculated as described previously (22).DNA isolation, bacterial transformation, and DNA sequenc-

ing. YCpMP2 DNA for bacterial transformation was re-leased from yeast cells by cellular disruption with glassbeads (14). Plasmid DNA was isolated from E. coli byalkaline extraction (33). Bacterial cells were transformed bya modification of the CaCl2 method (39). Mutant SUP4-ogenes were sequenced by dideoxynucleotide-mediated chaintermination on linearized double-stranded YCpMP2 mole-cules (23).

RESULTS

Plasmid retention and isolation of canavanine-resistant mu-tants. Cultures of MKP-op (RADI) and KAM1-p (radl),grown from low-titer inocula, were plated to measure plas-mid stability and to select canavanine-resistant mutants. Todetermine plasmid stability, the number of colonies thatformed on medium selective for the plasmid was comparedwith the corresponding value for nonselective medium.Approximately 88% of the MKP-op (37,299/42,231 = 0.88)and KAM1-p (9,968/11,513 = 0.87) cells retained YCpMP2.Since this value is typical for maintenance of YCpMP2 inMKP-op (11, 22, 39), the results indicate that the excisionrepair deficiency did not influence plasmid maintenance. Thefrequencies of all three detectable types of canavanine-resistant mutant were increased in the radl strain (Table 1).Presumably, the white or red and Lys' mutants were due toalterations at the CAN] locus or antisuppressor loci, respec-tively. Slightly more of the total canavanine-resistant mu-tants derived from KAMl-p than MKP-op were classified as

J. BACTERIOL.

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

SPECIFICITY OF YEAST radl MUTATOR 3011

TABLE 2. Sequence alterations in SUP4-o mutants

MKP-op (RADI) KAM1-p (radl)DNA sequence No No.

alteration detected Fr(10ency detected (10)7n(% of total) (% of total)

SubstitutionSingle change 262 (81.4) 16.4 211 (84.7) 111.0Tandem double 1 (0.4) 0.5Nontandem double 2 (0.6) 0.1 1 (0.4) 0.5

Deletion1 bp 21 (6.5) 1.4 16 (6.4) 8.5>1 bp 7 (2.2) 0.4 2 (0.8) 1.1

Insertion1 bp 1 (0.3) 0.06>1 bp 1 (0.4) 0.5Ty 25 (7.8) 1.6 17 (6.9) 8.9

Duplication 1 (0.3) 0.06Complex event 3 (0.9) 0.2Total 322 20.12 249 131.0

having SUP4-o mutations (red and Lys-). The mean fre-quency of spontaneous mutation at the SUP4-o locus was6.5-fold greater for KAMl-p than MKP-op, a significantincrease (P < 0.001 for a chi-square test comparing thenumbers of SUP4-o mutants and nonmutants among thetotal numbers of viable plasmid-bearing cells plated to selectcanavanine-resistant mutants for each strain). The corre-sponding mutation rates were calculated to be 9.1 x 10-7and 1.3 x 10-7 events per generation. It is clear that thedifferences observed between the repair-proficient and-deficient strain reflect the radi mutator effect rather thanvariation in their genetic backgrounds, since the strains wereisogenic except for the RADI locus.

Classification of SUP4-o mutants. To ensure the indepen-dence of the spontaneous mutations characterized by DNAsequencing, each mutant was obtained from a separateculture. Collections of 249 mutants arising in the radl strainand 322 mutants selected in the RADl wild-type backgroundwere analyzed (the results for 100 mutants isolated here inMKP-op were pooled with the data for 222 mutants exam-ined previously [22]). Between the two strains, 10 differentmutational classes were identified (Table 2). With the excep-tion of multiple-base-pair deletion, the relative proportionsof those classes of mutation common to both strains (singleand nontandem double-base-pair substitutions, single-base-pair deletion, and insertion of the 6-kb yeast retrotransposonTy) were similar. The increases in the frequencies of thesingle-base-pair events and Ty insertions in KAM1-p wereall approximately sixfold and together accounted for >98%of the overall radl mutator effect.

Analysis of base pair substitutions. The increment in thefrequency of single-base-pair substitution was responsiblefor 85% of the mutation frequency increase in the radl strain(Table 2). Both types of transition and all four possibletransversions were recovered in MKP-op and KAM1-p,although there was a small excess of transversions for eachstrain (Table 3). The relative fractions of the different basepair changes in the two strains were quite similar.The distributions, within SUP4-o, of the base pair substi-

tutions arising in MKP-op and KAM1-p, including doubleevents, are given in Fig. 1. A total of 67 sites were mutated,and some similarities were noted between the two distribu-tions. For both strains, base pair substitutions occurred atonly one position (site 51) within the tRNA intron, whereasthe remaining changes were distributed throughout the

TABLE 3. Relative fractions and frequenciesof base pair substitutions

MKP-op (RADI) KAMl-p (radi)Substitution No. detected Frequency No. detected Frequency

(% of total) (10-7) (% of total) (10-7)

TransitionsG C-* A T 69 (25.9) 4.3 53 (24.7) 27.7A T- G *C 41 (15.4) 2.5 14 (6.5) 7.3

Total 110 (41.3) 6.8 67 (31.2) 35.0TransversionsG C-T A 88 (33.1) 5.5 74 (34.4) 38.5G C- C G 52 (19.6) 3.2 56 (26.0) 29.1A T- C *G 5 (1.9) 0.3 7 (3.3) 3.7A*T. T A 11 (4.1) 0.7 11 (5.1) 5.7

Total 156 (58.7) 9.7 148 (68.8) 77.0

SUP4-o gene with 47 sites in common. In addition, two ofthe most frequently mutated sites (18 and 88) coincided, andof the specific substitutions at the common sites, 75% ofthose detected in MKP-op were also found in KAM1-p. Onthe other hand, a number of differences were also observed.Whereas no changes were recovered 5' or 3' to the SUP4-ocoding region in MKP-op, an A. T -- T. A transversionoccurring as part of a nontandem double event was detectedat position -2 in KAM1-p. This is the first substitution thatwe have identified outside of the region encoding the tRNA.However, since this change has not yet been detected as anindividual event among more than 3,400 SUP4-o mutationscharacterized to date, it might not reduce SUP4-o expres-sion or tRNA processing or function sufficiently to permit itsrecovery as a single substitution in our system. Base pairchanges were detected at fewer sites for KAMl-p than forMKP-op (50 versus 64), and the substitution frequencies inthe radl strain were from 3- to 52-fold greater at 42 of the 47common sites. Finally, 25% of the specific substitutionsdetected in the radl background at sites common to bothstrains were missing from the corresponding sites in theRADI distribution. By using the Monte Carlo estimate of theP value of the hypergeometric test (1) to compare the twodistributions statistically, we found that the probability ofrandom sampling error being the cause of differences in thetwo distributions was less than 1% (with 1,500 simulatedcomparisons, the P value was estimated to be 0.002 and the90% confidence interval ranged from 0.0005 to 0.0052).

Single-base-pair deletions and Ty insertions. Deletions ofsingle base pairs and insertions of the 6-kb retrotransposonTy into SUP4-o each accounted for 6.5% of the mutationfrequency increase attributable to the radl mutator effect(Table 2). In both strains, the majority (>93%) of deletionsoccurred in runs of two or more G C pairs and the numberof deletions increased with the number of base pairs in therun (Table 4), suggesting that their formation may haveinvolved strand slippage events (46). The run of five G. Cpairs located at positions 79 through 83 constituted a deletionhot spot in each strain. Ty insertions were detected at fourdifferent locations in SUP4-o (Table 4). Two or more Tyelements integrated at two sites in each strain, and more than80% of the total insertions were immediately 5' to position38, the SUP4-o site that we previously reported to be afrequent target for transposition (11). Recently, it has beendetermined that transposition of Ty elements into the URA3and LYS2 loci is also not random (36).Other mutational classes. A multiple-base-pair deletion, a

single-base-pair insertion, and a duplication were recoveredin MKP-op in this study. The deletion involved the loss of 27

VOL . 172, 1990

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

3012 KUNZ ET AL.

TTG

T GT AT AC AA TATA CAAA T AAAG TAGA AAAGCT

I 1-2 1 10

3' AT GAGAGCCATC

T A AAGC AA AGGG TA TG G TT TGT TTTT

GTTAAAAAAAAA

cccc2

C AAAA AA

AAAAAAAATAA

T CT CTT CAGTACCAGCAC

CCAGAACG

cA

T AT AT A CG AG GC

GGCAG GCCAACAGTGCA

AAAAAA TA TA G T TA C A TA C A AA C A AA C A AA CTACCAC

20 30 40 50 60GGTTCAACCA AATTCCGCGT TCTCAAATTA AATAGTGATG CTTTAGMCT

AC A MAAC A AACC G AA

G AAAAAGATAAAAAA

A AAAACACMCTTA CTTA C2C C

cccT 2T TTTTTT

CA AC G CG A GCA GTA CTATTTT

A CTATAA TTT GA TT GA T GG TG TT T

TT

C TC G TC G GTTC GTACT

TC CAGACA

GGGGACAC

cccA CTCAGCC

TTT TTT TTT TTT TTT TCG TCG TCCG TCCA T TCCA T TAAAGG TAAAGG AA

TTMGGAAA

70 80 89CTAGCCCCCA AGCTGAGCGG GGGCCCTCT 5'

C AAAGCGCC AA GC

CA TCA TTATGGGGGGGGT

CGA CATGA TATGCTGC

G

T C CA A MT C TT G ACT C T G ACT T G

T TT T

TTTTTT

FIG. 1. Distribution of spontaneous base pair substitutions in the SUP4-o gene. For simplicity, only the region of the transcribed strandencoding the tRNA is shown, along with the first two upstream bases. The anticodon is at 36 to 38 and the 14-base-pair intron extends from40 through 53 (18). Mutations isolated in MKP-op (RADI) and KAMl-p (radl) are presented above and below the transcribed strand,respectively. Numbered substitutions indicate changes in double mutants.

base pairs from positions 63 through 89, the insertion oc-curred in the run of 5 G- C pairs at positions 79 through 83,and the duplication was a direct repeat of the 7-base-pairsequence encompassing positions 72 through 78. The othermultiple-base-pair deletions and the complex events identi-fied in MKP-op have been detailed elsewhere (11). Twotriple-base-pair deletions (77 through 79) and a double-

TABLE 4. Characterization of single-base-pair deletionsand Ty insertions

MKP-op (RADI) KAM1-p (radl)

Event Sitea No. No.detected Frequency detected Frequency

(% of total) (% of total) (1)

Deletion 15 1 (4.8) 0.6718 or 19 1 (4.8) 0.6725 or 26 1 (6.2) 5.3

29 1 (6.2) 5.365 - 67 4 (25.0) 21.2

72 1 (4.8) 0.6779-83 16 (76.2) 10.7 8 (50.0) 42.584-86 2 (9.4) 1.3 2 (12.6) 10.7

Total 21 14.01 16 85.0

Insertion 6 + 7 3 (17.6) 15.717 + 18 2 (8.0) 1.337 + 38 22 (88.0) 14.1 14 (82.4) 73.343 4* 44 1 (4.0) 0.64

Total 25 16.04 17 89.0a For deletions in runs of base pairs, the position of the run is given since

the precise base pair eliminated cannot be determined. Ty insertions wereidentified as described previously (11); the symbol * indicates that Ty ispresumed to have inserted between the two sites.

base-pair insertion (5'-GC-3' found between positions 64 and65) were detected in the radl strain.

DISCUSSIONDisruption of the RADI gene clearly resulted in a mutator

phenotype. However, there has been controversy regardingthe ability of radl defects to enhance spontaneous mutagen-esis (43). Previously, detection of the mutator effect appar-ently depended on the assay system and the particular radlallele used, suggesting that the mutator phenotype might beobscured by its own specificity or by leakiness of individualradl alleles. Since the frequencies of all types of base pairsubstitution were increased in the radl strain, it is doubtfulthat the radl mutator effect could be concealed by itsspecificity alone. However, base pair changes were notdetected at all sites in SUP4-o where substitutions occurredin the isogenic RADI strain. Thus, neighboring DNA se-quences might modulate the radl mutator effect at specificbase pairs. If so, this could explain why radl strains werenot always found to be mutators when mutation was assayedonly at specific sites. Leakiness of the radl alleles usedmight also have been a complicating factor.

Increases in the frequencies of single-base-pair substitu-tion, single-base-pair deletion, and Ty insertion accountedfor almost the entire change in the SUP4-o mutation fre-quency in the radl strain. The relative fractions of theseevents were essentially the same in both strains, and theirfrequencies were increased to similar extents in KAM1-p,indicating that the three classes of mutation were coordi-nately enhanced by the radl mutator effect. Since RADIdefects are believed to cause excision repair deficiency, it isreasonable to suppose that unexcised spontaneous damage,which normally would be removed by excision repair, could

J. BACTERIOL.

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

SPECIFICITY OF YEAST radl MUTATOR 3013

play a key part in this enhancement. Given that single-base-pair events and transposition of Ty elements werespecifically promoted in the radl strain, it seems probablethat if unrepaired spontaneous DNA lesion are involved,then at least two different mechanisms would have to beresponsible for the radl mutator phenotype.

Like radl defects, rad3 mutations that increase UV sen-sitivity appear to eliminate the incision step of excisionrepair (9). It has been shown that the mutator phenotypeassociated with the rad3-12 allele, which markedly enhancesUV sensitivity (42) and so is unlikely to be a remi allele ofRAD3 (9), can be offset by the antimutator effect conferredby defective alleles of the REV3 gene (42). In fact, rad3-12rev3 double mutants were found to have reduced spontane-ous mutation rates similar to those of rev3 antimutatorstrains alone (42). Recently, DNA sequencing of the REV3gene has led to the prediction that it encodes a novel,nonessential DNA polymerase (34). Since REV3 is requiredfor mutagenesis by UV, 4-nitroquinoline-1-oxide and gammarays but is distinct from the genes encoding the three knownyeast nuclear DNA polymerases, it was concluded that theREV3 polymerase likely functions only in translesion syn-thesis (34). If so, then translesion synthesis should becapable of producing single-base-pair substitutions and de-letions because UV induction of these events is substantiallydecreased by defects in REV3 (28, 29). Here we havedetermined that the radl mutator enhanced the frequenciesof spontaneous single-base-pair substitutions and deletions.Taken collectively, these findings suggest that the radlmutator phenotype in yeast cells could be explained, in largepart, by error-prone translesion synthesis across spontane-ous damage, which is a substrate for excision repair. Thenotion of translesion synthesis past unrepaired spontaneousdamage is reminiscent of the hypothesis that spontaneouslesions are channeled through mutagenic repair pathways instrains deficient in error-free repair (12, 42). The difference isthat in the case of translesion synthesis, elevated spontane-ous mutagenesis would not reflect mutagenic repair ofDNAdamage but rather tolerance of spontaneous DNA lesions viaan error-prone mechanism.

Insertion of the yeast retrotransposon Ty into SUP4-o wasalso increased in the radl background. Elsewhere, we havedemonstrated that Ty transposition is severely reduced in aradS2 strain (22) but is not influenced by disruption ofRAD18 (B. A. Kunz et al., submitted for publication). It hasbeen found that treatment with UV or 4-nitroquinoline-1-oxide, mutagens that produce DNA damage which issubject to excision repair, stimulates transcription of Tyelements and activates their transposition (4, 31). Althoughthis activation is mutagen dose dependent and so presum-ably correlates with the production of DNA damage, theprecise mechanism(s) at work has not yet been elucidated.Nevertheless, these results plus our findings argue thatactivation ofTy transposition by spontaneous DNA damage,which would usually be repaired in a RADI strain, contrib-utes to the radl mutator phenotype. After this report wassubmitted for publication, it was reported that mutations inRAD6, a yeast gene whose product polyubiquitinates his-tones in vitro, increase Ty transposition into the CAN] andURA3 genes (38).

It should also be considered that the radl mutator effectcould be unrelated to spontaneous damage (26, 43). Onepossible explanation would be that defects in the RADI genemight somehow reduce the accuracy of DNA synthesis.Since Ty retrotransposition is increased in the radl strain, itseems improbable that altered replicational fidelity alone

could be responsible for the mutator phenotype. Still, itcould be that a decrease in the fidelity of replicative DNAsynthesis is responsible for the enhanced frequencies ofsingle-base-pair substitution and deletion in the radl strain.However, this would be difficult to reconcile with the abilityof rev3 alleles to eliminate the rad3-12 mutator phenotype,since the predicted REV3 polymerase is not required forreplicative DNA synthesis (34).

Alternatively, it has been suggested that excision repairfunctions might recognize a subset of DNA replicationerrors, presumably mismatches (32). Accordingly, the in-crease in single-base-pair deletions and substitutions in theradl strain might reflect a deficiency in mismatch correction.In yeast cells, a frameshift mismatch (single-nucleotide loopopposite a missing nucleotide) can be repaired to restore themissing information or fix the deletion (3, 19). Furthermore,in one study (19), reductions in mismatch repair were foundto enhance the incidence of deletion. Thus, a defect in repairof frameshift mismatches could account for the increaseddeletion frequency in the radl background. On the otherhand, it seems less likely that disruption of the RADI geneenhanced substitution mutagenesis by perturbing mismatchcorrection. There was no significant increase in the fractionof a particular type(s) of base-pair change in the radl strain,arguing that the specificity of mismatch correction was notaltered. If instead the RADI disruption reduced the overallefficiency of mismatch correction, an increase in transitionswould be expected in the radl background, since yeastmismatch repair corrects transition mismatches more effi-ciently than most transversion mismatches (3, 19), yet theratio of transversions to transitions was slightly greater inKAM1-p than in MKP-op.

In conclusion, the radl mutator effect in yeast cellsspecifically increases the frequencies of single-base-pairsubstitution, single-base-pair deletion, and Ty insertion. Wecannot unequivocally discount the possibilities that disrup-tion of RADI enhanced single-base-pair events by decreas-ing the fidelity of replicative DNA synthesis or the efficiencyof mismatch correction. However, the simplest explanationwould be that the mutator phenotype involves error-pronetranslesion synthesis past unrepaired spontaneous damageand activation of Ty transposition by such damage.

ACKNOWLEDGMENTS

We thank W. T. Adams for running the computer analysis of thebase pair distributions, R. L. Klein for supplying plasmid pL%2,and M. Gabriel, T. Kolodka, and F. Yadao for technical assistance.T.K. was supported by a Natural Sciences and Engineering

Research Council of Canada undergraduate scholarship, and M.G.and F.Y. were supported in part by a subsidy from the ManitobaCareerstart '89 Program. This work was supported by the NaturalSciences and Engineering Research Council of Canada.

LITERATURE CITED1. Adams, W. T., and T. R. Skopek. 1987. Statistical test for the

comparison of samples from mutational spectra. J. Mol. Biol.194:391-396.

2. Aguilera, A., and H. L. Klein. 1989. Yeast intrachromosomalrecombination: long gene conversion tracts are preferentiallyassociated with reciprocal exchange and require the RADI andRAD3 gene products. Genetics 123:683-694.

3. Bishop, D. K., J. Andersen, and R. D. Kolodner. 1989. Speci-ficity of mismatch repair following transformation of Saccharo-myces cerevisiae with heteroduplex plasmid DNA. Proc. Natl.Acad. Sci. USA 86:3713-3717.

4. Bradshaw, V. A., and K. McEntee. 1989. DNA damage activatestranscription and transposition of yeast Ty retrotransposons.

VOL. 172, 1990)

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

3014 KUNZ ET AL.

Mol. Gen. Genet. 218:465-474.5. Brendel, M., N. A. Khan, and R. H. Haynes. 1970. Common

steps in the repair of alkylation and radiation damage in yeast.Mol. Gen. Genet. 106:289-295.

6. Chanet, R., C. Izard, and E. Moustacchi. 1976. Genetic effectsof formaldehyde in yeast. III. Influence of ploidy and mutationsaffecting radiosensitivity on its lethal effects. Mutat. Res. 35:29-38.

7. Chanet, R., N. Magana-Schwencke, and F. Fabre. 1988. Poten-tial DNA-binding domains in the RAD18 gene product ofSaccharomyces cerevisiae. Gene 74:543-547.

8. Cooper, A. J., and R. Waters. 1987. A complex pattern ofsensitivity to simple monofunctional alkylating agents amongstthe rad mutants. Mol. Gen. Genet. 209:142-148.

9. Friedberg, E. C. 1988. Deoxyribonucleic acid repair in Saccha-romyces cerevisiae. Microbiol. Rev. 52:70-102.

10. Game, J. C., and B. S. Cox. 1973. Synergistic interactionsbetween rad mutations in yeast. Mutat. Res. 20:35-44.

11. Giroux, C. N., J. R. A. Mis, M. K. Pierce, S. E. Kohalmi, andB. A. Kunz. 1988. DNA sequence analysis of spontaneousmutations in the SUP4-o gene of Saccharomyces cerevisiae.Mol. Cell. Biol. 8:978-981.

12. Hastings, P. J., S.-K. Quah, and R. C. von Borstel. 1976.Spontaneous mutation by mutagenic repair of spontaneouslesions in DNA. Nature (London) 264:719-722.

13. Hoekstra, M. F., and R. E. Malone. 1986. Excision repairfunctions in Saccharomyces cerevisiae recognize and repairmethylation of adenine by the Escherichia coli dam gene. Mol.Cell. Biol. 6:3555-3558.

14. Hoffman, C. S., and F. Winston. 1987. A ten-minute DNApreparation from yeast efficiently releases autonomous plasmidsfor transformation of Escherichia coli. Gene 57:267-272.

15. Ito, Y., Y. Fukada, K. Murata, and A. Kimura. 1983. Transfor-mation of intact yeast cells treated with alkali ions. J. Bacteriol.153:163-168.

16. Jones, J. S., S. Weber, and L. Prakash. 1988. The Saccharomycescerevisiae RAD18 gene encodes a protein that contains potentialzinc finger domains for nucleic acid binding and a putative nucle-otide binding sequence. Nucleic Acids Res. 16:7119-7131.

17. Klein, H. 1988. Different types of recombination events arecontrolled by the RADI and RADS2 genes of Saccharomycescerevisiae. Genetics 120:367-377.

18. Knapp, G., J. S. Beckwith, P. F. Johnson, S. A. Fuhrman, and J.Abelson. 1978. Transcription and processing of interveningsequences in yeast tRNA genes. Cell 14:221-236.

19. Kramer, B., W. Kramer, M. S. Williamson, and S. Fogel. 1989.Heteroduplex DNA correction in Saccharomyces cerevisiae ismismatch specific and requires functional PMS genes. Mol.Cell. Biol. 9:4432-4440.

20. Kunz, B. A., J. D. Armstrong, M. Glattke, S. E. Kohahmi, andJ. R. A. Mis. 1990. The SUP4-o system for analysis of muta-tional specificity in yeast, p. 337-346. In M. L. Mendelsohn andR. J. Albertini (ed.), Fifth international conference on environ-mental mutagens. Alan R. Liss, Inc., New York.

21. Kunz, B. A., F. Eckardt, and R. H. Haynes. 1985. Analysis ofnon-linearities in frequency curves for UV-induced mitoticrecombination in wild-type and excision-repair-deficient strainsof yeast. Mutat. Res. 151:235-242.

22. Kunz, B. A., M. G. Peters, S. E. Kohlami, J. D. Armstrong, M.Glattke, and K. Badiani. 1989. Disruption of the RAD52 genealters the spectrum of spontaneous SUP4-o mutations in Sac-charomyces cerevisiae. Genetics 122:535-542.

23. Kunz, B. A., M. K. Pierce, J. R. A. Mis, and C. N. Giroux. 1987.DNA sequence analysis of the mutational specificity of u.v. lightin the SUP4-o gene of yeast. Mutagenesis 2:445-453.

24. Kurjan, J., and B. D. Hail. 1982. Mutations at the Saccharomy-ces cerevisiae SUP4 tRNATYr locus: isolation, genetic finestructure mapping, and correlation with physical structure. Mol.Cell. Biol. 2:1501-1513.

25. Kurjan, J., B. D. Hall, S. Gilliam, and M. Smith. 1980. Muta-tions at the yeast SUP4 tRNATYr locus: DNA sequence changesin mutants lacking suppressor activity. Cell 20:701-709.

26. Lawrence, C. W. 1982. Mutagenesis in Saccharomyces cerevi-siae. Adv. Genet. 21:173-254.

27. Lawrence, C. W., and R. B. Christensen. 1976. UV mutagenesisin radiation sensitive strains of yeast. Genetics 82:207-232.

28. Lawrence, C. W., and R. B. Christensen. 1979. Ultraviolet-induced reversion of cycl alleles in radiation-sensitive strains ofyeast. Genetics 92:397-408.

29. Lawrence, C. W., T. O'Brien, and J. Bond. 1984. Ultraviolet-induced reversion of his4 frameshift mutations in rad6, revi,and rev3 mutants of yeast. Mol. Gen. Genet. 195:487-490.

30. McCready, S. J., and B. S. Cox. 1973. Antisuppressors in yeast.Mol. Gen. Genet. 124:305-320.

31. McEntee, K., and V. A. Bradshaw. 1988. Effects of DNAdamage on transcription and transposition of Ty retrotrans-posons of yeast, p. 245-253. In M. E. Lambert, J. F. McDonald,and I. B. Weinstein (ed.), Eukaryotic transposable elements asmutagenic agents. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

32. Monteleone, B. A., M. F. Hoekstra, and R. E. Malone. 1988.Spontaneous mitotic recombination in yeast: the hyper-recom-binational remi mutations are alleles of the RAD3 gene. Genet-ics 119:289-301.

33. Moreile, G. 1989. A plasmid extraction procedure on a miniprepscale. Focus 11:7-8.

34. Morrison, A., R. B. Christensen, J. Alley, A. K. Beck, E. G.Bernstein, J. F. Lemontt, and C. W. Lawrence. 1989. REV3, aSaccharomyces cerevisiae gene whose function is required forinduced mutagenesis, is predicted to encode a nonessentialDNA polymerase. J. Bacteriol. 171:5659-5667.

35. Moustacchi, E. 1969. Cytoplasmic and nuclear genetic eventsinduced by UV light in strains of Saccharomyces cerevisiaewith different UV sensitivities. Mutat. Res. 7:171-185.

36. Natsoulis, G., W. Thomas, M.-C. Roghmann, F. Winston, andJ. D. Boeke. 1989. Tyl transposition in Saccharomyces cerevi-siae is nonrandom. Genetics 123:269-279.

37. Newlon, C. S. 1988. Yeast chromosome replication and segre-gation. Microbiol. Rev. 52:568-601.

38. Picologlou, S., N. Brown, and S. W. Liebman. 1990. Mutations inRAD6, a yeast gene encoding a ubiquitin-conjugating enzyme,stimulate retrotransposition. Mol. Cell. Biol. 10:1017-1022.

39. Pierce, M. K., C. N. Giroux, and B. A. Kunz. 1987. Develop-ment of a yeast system to assay mutational specificity. Mutat.Res. 182:65-74.

40. Prakash, L. 1976. Effects of genes controlling radiation sensi-tivity on chemically induced mutations in Saccharomyces cere-visiae. Genetics 83:285-301.

41. Prakash, L., and. S. Prakash. 1977. Isolation and characteriza-tion of mms-sensitive mutants of Saccharomyces cerevisiae.Genetics 86:33-55.

42. Quah, S.-K., R. C. von Borstel, and P. J. Hastings. 1980. Theorigin of spontaneous mutation in Saccharomyces cerevisiae.Genetics 96:819-839.

43. Sargentini, N. J., and K. C. Smith. 1985. Spontaneous mutagen-esis: the roles of DNA repair, replication, and recombination.Mutat. Res. 154:1-27.

44. Schiestl, R. H., and S. Prakash. 1988. RADI, an excision repairgene of Saccharomyces cerevisiae, is also involved in recombi-nation. Mol. Cell. Biol. 8:3619-3626.

45. Snow, R. 1968. Recombination in ultraviolet sensitive strains ofSaccharomyces cerevisiae. Mutat. Res. 6:409-418.

46. Streisinger, G., Y. Okada, J. Emrich, J. Newton, A. Tsugita, E.Terzaghi, and M. Inouye. 1966. Frameshift mutations and thegenetic code. Cold Spring Harbor Symp. Quant. Biol. 31:157-179.

47. von Borstel, R. C., and P. J. Hastings. 1980. DNA repair andmutagen interactions in Saccharomyces: theoretical consider-ations, p. 159-167. In W. M. Generoso, M. D. Shelby, and F. J.de Serres (ed.), DNA repair and mutagenesis in eukaryotes.Plenum Publishing Corp., New York.

48. Wang, S. S., and A. K. Hopper. 1988. Isolation of a yeast geneinvolved in species-specific tRNA processing. Mol. Cell. Biol.8:5140-5149.

49. Zuk, J., D. Zabrowska, and Z. Swietlinska. 1979. Comparison ofsensitivity and liquid holding recovery in rad mutants of Sac-charomyces cerevisiae inactivated by UV and DEB. Mol. Gen.Genet. 166:91-96.

J. BACTERIOL.

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from