Mutations in RAD3, MSH2, and RAD52 Affect the Rate ofGene Amplification in the Yeast Saccharomyces cerevisiae

Christopher Peterson, Jennifer Kordich, Laura Milligan, Erika Bodor,Angela Siner, Kristin Nagy, and Charlotte Elder Paquin*

Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio

We report here the use of the ADH4:CUP1 ampli-fication detection system to identify five high am-plification rate (HAR) strains of Saccharomyces cer-evisiae that display 40- to 600-fold higheramplification rates than those of parental strains.We have identified a mutation in RAD3 DNA re-pair helicase gene in HAR strain B9-40 that resultsin a 40-fold increase in amplification rate. RAD3 isthe functional homolog of the human XPD gene,suggesting that this model system will provide im-portant candidates for genes that affect gene am-plification in human cells. Isolation of the HAR

strains has allowed us to test whether RAD52,which is essential for recombinational repair ofDNA double-strand breaks, is also essential foramplification. Deletion of RAD52 in HAR strainsB3-10 and B11-60 decreases amplification ap-proximately 100-fold. In contrast, deletion ofMSH2, which increases recombination betweensequences with limited similarity, increases the am-plification rate about 10-fold. These results suggestthat recombination is an important step in amplifi-cation. Environ. Mol. Mutagen. 36:325–334,2000. © 2000 Wiley-Liss, Inc.

Key words: recombination; gene amplification; RAD3; RAD52; MSH2

INTRODUCTION

Primary gene amplification, the mutation from one genecopy per genome to two or more copies per genome, is amajor mechanism underlying oncogene overexpression inhuman cancers [Bishop, 1991; Lengauer et al., 1991].Clearly, an understanding of gene amplification mecha-nism(s) is essential to a complete understanding of carcino-genesis. Intensive investigation of gene amplification struc-tures in mammalian cells has led to the proposal of a widerange of models to explain the mechanism(s) of gene am-plification. In general, these models can be classified asinvolving errors in replication, recombination, or the repairof broken chromosomes, and can involve a single step ormultiple steps in the formation of the amplified DNA [re-viewed in Stark et al., 1989; Windle et al., 1991; Stark,1993]. However, it has proven difficult to test these modelsexperimentally and to demonstrate whether the amplifica-tion structures represent the initial amplification event orsubsequently stabilized structures.

Gene amplification is undetectable in normal mammaliancells (frequency, 1 3 1029 amplifications per cell) [Tlsty,1990; Wright et al., 1990], but tumorigenic cells have highamplification frequencies (13 1025 to 1 3 1023 amplifi-cations per cell) [Sager et al., 1985; Otto et al., 1989; Tlstyet al., 1989]. This suggests that normal mammalian cellshave a mechanism(s) to suppress amplification. The firstmammalian gene shown to affect gene amplification,p53,was tested because it had already been shown to be a tumorsuppressor and is the most common genetic alteration foundin sporadic, nonfamilial cancers [Livingstone et al., 1992;

Yin et al., 1992]. However,p53 is not the only gene thataffects amplification potential, as some tumor cells express-ing wild-type p53 still have high gene amplification fre-quencies [Livingstone et al., 1992]. In an effort to identifysuch genes in mammalian cells, four hamster cell lines thatshow an increased ability to amplify endogenous geneswere isolated [Giulotto et al., 1987] and subsequentlyshown to be UV sensitive [Giulotto et al., 1991]. Althoughthe defect causing the amplificator phenotype in these cellsremains unclear, analysis of cell lines representing fourknown UV-sensitive, excision repair–deficient complemen-tation groups indicated that cells with mutantERCC6(ex-cision repair cross-complementing group 6, a gene that

Grant sponsor: National Cancer Institute–Frederick Cancer Research andDevelopment Program; Grant sponsor: American Cancer Society; Grantnumber: VM-117A; Grant sponsor: National Institutes of Health; Grantnumber: P30 ES06096.

C. Peterson and J. Kordich are currently at University of Cincinnati,College of Medicine, Department of Molecular Genetics, Biochemistry,and Microbiology, Cincinnati, OH 45221.

A. Siner is currently at University of Cincinnati, College of Medicine,Department of Environmental Health, Cincinnati, OH 45221.

K. Nagy is currently at University of Michigan, School of Education, AnnArbor, Michigan.

*Correspondence to: Charlotte Paquin, University of Cincinnati, Depart-ment of Biological Sciences, P.O. Box 210006, Cincinnati, OH 45221-0006. E-mail: paquincm@email.uc.edu

Received 7 October 1999; provisionally accepted 11 November 1999; andin final form 26 August 2000

Environmental and Molecular Mutagenesis 36:325–334 (2000)

© 2000 Wiley-Liss, Inc.

encodes a DNA helicase involved in the preferential repairof transcribed sequences) displayed a fourfold increase inamplification over that of control cells [Mondello et al.,1995].

Our lab has developed a two-step system to phenotypi-cally detect coamplifications of theADH4 andCUP1genesin the yeastSaccharomyces cerevisiae[Dorsey et al., 1993].The amplification rate in wild-type strains is low (;1 310210 amplifications/cell/generation) [Dorsey et al., 1992]but detectable with this system. The genetic and moleculartools available in yeast allow us to address mechanisticquestions that have been difficult to examine in mammaliancells.

One controversy in amplification is whether recombina-tion is an essential step in gene amplification. One recom-bination model, the intramolecular recombination model ofgene amplification [Butler et al., 1995, 1996], is describedbelow. We have chosen to examine this specific modelbecause it provides testable predictions and suggests amechanism by which both linear and circular extrachromo-somal amplifications are produced. Both types of amplifi-cations have been observed among the spontaneous ampli-fications we have isolated [Walton et al., 1986; Dorsey etal., 1992, 1993]. The first step in the model is the productionof a DNA double-strand break adjacent to inverted repeats.The second step is a recombination event between invertedrepeats to form a hairpin molecule. The third step is repli-cation of the hairpin. If the break is between the target geneand the centromere, replication results in a linear extrachro-mosomal amplification carrying two copies of the targetgene, similar to those previously characterized in our sys-tem. If the break occurs between the target gene and thetelomere, replication results in a dicentric chromosome car-rying two copies of the target gene, which initiates a break-age-fusion-bridge cycle [McClintock, 1984]. Such a break-age-fusion-bridge cycle could increase the number of copiesof the target gene and eventually be resolved as a circularamplification [Stark, 1993]. This model predicts that recom-bination is an essential step in the formation of both linearand circular extrachromosomal amplifications.

This intramolecular recombination model is based on theamplification of rDNA in Tetrahymena,but Butler et al.[1996] showed that such a mechanism can occur in yeast.They transformed yeast with a circular plasmid, inducedDNA double-strand breaks adjacent to 42-bp inverted re-peats, and recovered at a high rate linear extrachromosomalpalindromes similar toADH4:CUP1palindromes. In addi-tion, Butler et al. [1996] were the first to demonstrate thatpalindrome formation was dependent on the presence of thewild-typeRAD52gene in yeast. Although the exact functionof RAD52in recombination is not yet known, many studieshave demonstrated the importance ofRAD52in recombina-tion and the repair of DNA double-strand breaks [Petes etal., 1991]. Specifically, deletion ofRAD52has been shownto decrease recombination between inverted repeats 3000-

fold [Rattray et al., 1994]. Recent evidence suggests that theRAD52 protein promotes DNA strand annealing[Mortensen et al., 1996] and facilitates access of the strand-exchange protein pRAD51, to single-stranded DNA boundby the single-stranded binding protein RPA [Sung, 1997].

One important test of the intramolecular recombinationmodel is to determine whetherRAD52is also essential foramplification of genomic DNA without the artificial induc-tion of DNA double-strand breaks. The spontaneous ampli-fication rate is so low, however, that it is difficult to con-vincingly demonstrate that amplification rates are decreasedin normal cells. However, we report here the use of theADH4:CUP1system to isolate five independent strains ofS.cerevisiae,all of which display high amplification rates. Wehave identified the mutation that increases the amplificationrate in one high amplification rate (HAR) strain, as a mu-tation in the DNA repair helicase geneRAD3,which resultsin a 40-fold increase in the gene amplification rate. Thefunctional human homolog of theS. cerevisiae RAD3geneis XPD, one of the genes mutated in the cancer-susceptibil-ity syndrome, xeroderma pigmentosum [Sung et al., 1993].The HAR strains were also used to demonstrate that dele-tion of RAD52 decreased amplification in HAR strainsB3-10 and B11-60. HAR strains have also allowed us toisolate a large number of independent amplifications forstructural analysis. Thus, these HAR strains provide severalnew approaches to mechanistic analysis of amplification. Tofurther examine the role of recombination in amplificationwe tested the effect of anMSH2deletion on amplification.An MSH2deletion has been shown to increase the rate ofrecombination between similar, but not identical, DNA se-quences [Selva et al., 1995].

MATERIALS AND METHODS

Yeast Strains and Media

The yeast strains used in mutagenesis are 4-11B (MATa) and 4-6D(MATa). These haploids areadh1-D1, haveCUP1deleted from its normallocation, and contain theADH4:CUP1construct. The mating of these twostrains forms the previously described strain A4C [Dorsey et al., 1993]. Allthe HAR strains reported here were derived from haploid 4-11B aftermutagenesis. 4-11BOMT is aMATa strain isogenic to 4-11B, constructedby using a galactose-induced HO gene on a plasmid (gift from J. Strathern)to induce mating-type switching [Herskowitz and Jensen, 1991]. B9-40 2Nwas constructed by inducing mating-type switching in strain B9-40 usingthe HO plasmid and then mating the resultingMATa strain back to theparental MATa B9-40 strain. CCP8A is a haploidADH4:CUP1,RAD9-D::URA3 strain, constructed by crossing YJJ53, arad9-deletionstrain obtained from the Yeast Genetic Stock Center (Berkeley, CA), with4-11B, sporulating the resulting diploid and isolating anADH4:CUP1,rad9-D::URA3spore. Two such spores were mated to construct the diploidCCP8A 2N strain that is homozygous for theRAD9deletion andADH4:CUP1.

To delete theLEU2, RAD52,and MSH2 genes we used the standardyeast knockout strategy of transforming yeast carryingura3-52 mutation,with DNA in which the open reading frame of the gene to be deleted hadbeen replaced by theURA3gene, and selecting strains that could grow in

326 Peterson et al.

the absence of uracil [Rothstein, 1991]. In addition, theURA3 gene wasflanked by direct repeats, so that after deletion of the gene we could selectfor loss of theURA3gene on 5-fluroorotic acid (5-FOA; PCR, Gainesville,FL) to ensure that any differences between the amplification rates in thestrains resulted from the deletion ofRAD52or MSH2and not to whetherthe strains carriedURA3or ura3-52 alleles [Alani et al., 1987].

The plasmids used were theLEU2 gene blaster pNK85 [Alani et al.,1987] and theRAD52gene blaster plasmid pBDG542, which has theURA3gene. TheHISG direct repeats inserted into theBglII site in the RAD52ORF were a gift from Dwight Nissley and J. Strathern, and theMSH2blaster pSH149, which has theURA3 gene, and theHISG direct repeatsinserted between twoBamHI sites in theMSH2gene wasere a gift from S.Holbeck and J. Strathern. The replacement ofLEU2, MSH2,andRAD52byURA3was confirmed by Southern blot analysis.

The B9-40 and 4-11B isogenic strains, differing only at codon 463sequence, were constructed in strain B9-40Dleu2 and 4-11BOMTDleu2by standard gene replacement techniques [Rothstein, 1991], which utilizedthe pRS406:rad3-I463K(for strain 4-11BOMTleu2ura3) or pRS406:RAD3(for strain B9-40Dleu2) integrating vectors described below, linearized attheBglII site in the center of theRAD3open reading frame. Strains that hadlost the pRS406 plasmid and one of the twoRAD3 alleles were thenselected for by growth on 5-FOA.

The UV sensitivity and the codon 463 sequence of 5-FOA–resistantstrains were determined as previously described to identify theRAD3allelepresent in each strain. The media used are described in Dorsey et al. [1993]and Sherman [1991]. Tetrad analysis was done as described by Sherman[1991].

ADH4:CUP1 Amplification Detection System

TheADH4:CUP1amplification detection system described in Dorsey etal. [1993] was used to identify antimycin A–resistant, copper-resistantmutants (coamplifications ofADH4 andCUP1confer an antimycin A–re-sistant phenotype as a result of overexpression ofADH4 and a copper-resistant phenotype as a result of overexpression ofCUP1), both todetermine whether these mutants carry amplifications using Southern blotanalysis of chromosomal DNA separated on pulsed-field gels and toestimate amplification rates.

Mutagenesis and HAR Screen

Strains 4-11B and 4-6D were mutagenized withN-methyl-N9-nitro-N-nitrosoguanidine (MNNG; Sigma, St. Louis, MO) by standard techniques

[Lawrence, 1991]. Mutagenized 4-11B and 4-6D cells (viability, 5%)were plated to single cells on YEP glucose medium to isolate independentcandidate HAR strains. Since theADH4:CUP1 amplification rate in theparental strain is normally 13 10210 amplifications/cell/generation (TableI), we wanted to allow enough cell divisions to detect a 10- to 100-foldincrease in amplification rate. Therefore, colonies were grown at 30°C toapproximately 13 108 cells. A high frequency of cells containing ampli-fied ADH4:CUP1DNA that arose during colony formation would indicatea potential HAR strain and was determined in the following steps: 4758mutagenized colonies were picked, suspended in sterile water, streaked toa new YEP glucose plate for retrieval of the original strain, then plated toantimycin A medium. The frequency of antimycin A mutants for eachstrain was determined by plating the remainder of the original colony toantimycin A medium. Early observations indicated that most coloniesderived from mutagenizedADH4:CUP1cells displayed a frequency of lessthan 10 antimycin A–resistant mutants. We thus considered this frequencyto be background and three times this frequency (30 antimycin A–resistantmutants) to be the criterion for candidate HAR strains (250 mutagenizedstrains met this criteria). Antimycin A–resistant mutants were restreaked onantimycin A media and allowed to grow an additional 3 to 5 days beforedetermination of copper resistance.

It is important to note that antimycin A–resistant mutants derived froma single strain often had different growth rates, suggesting they may havearisen from separate mutations. Often hundreds of antimycin A–resistantmutants were derived from a single candidate strain, so we were careful toscreen a random sample of 25 to 30 mutants encompassing all antimycinA–resistant growth phenotypes for copper resistance. Copper resistance ofantimycin A–resistant mutants was measured as described by Dorsey et al.[1993] on complete media or complete media containing 0.75 or 1.0 mMcopper sulfate. Sixty-six strains had at least one antimycin A–resistantmutant that was also copper resistant. All antimycin A, copper-resistantmutants were then analyzed forADH4:CUP1 amplification by Southernblot analysis of chromosome preparations separated on pulsed-field gels.These strains are haploid and therefore contain only one copy of theADH4:CUP1cassette on chromosomeVII. Thus for this study, we definedan amplification as an antimycin A, copper-resistant mutant containing anew, second band hybridizing theADH4:CUP1probe in Southern blots ofchromosomal DNA separated on pulsed-field gels (examples shown in Fig.1). This screen, however, would not allow us to identify intrachromosomalamplifications that do not significantly alter the size of chromosomeVII.Thirty-eight of the candidate HAR strains had at least one antimycinA–resistant, copper-resistant mutant that carried an amplification of theADH4:CUP1cassette.

TABLE I. Amplification Rates of HAR Strains

StrainNumber ofcells/culture

Number ofantimycinA–resistant

mutantsscreened on

copper

Percentage ofantimycin A,

copper-resistantmutants withADH4:CUP1amplification

Number ofindependent

amplifications/total numberof culturesa

Amplificationrate estimateb

4-11B 3.33 108 680 33 (2/6) 2/120 56 8 3 10211

4-6D 2.13 108 300 100 (4/4) 4/60 36 4 3 10210

Combinedc 2.93 108 980 60 (6/10) 6/180 16 1 3 10210

B3-10 1.43 108 936 99 (84/85) 28/38 16 0.43 1028

B9-40 1.63 108 1000 18 (27/148) 22/50 46 2 3 1029

B11-60 2.13 108 1200 59 (143/242) 44/60 66 2 3 1029

B12-123 1.63 107 1238 4 (11/269) 10/59 16 0.73 1028

B12-267 2.93 107 1200 38 (115/306) 49/60 66 2 3 1028

aAmplifications determined by Southern blot analysis of chromosomes separated on pulsed-field gels.bAmplification rate estimates and 95% confidence intervals calculated by the P0 method of Lea and Coulson [1949].cData are a compilation of the two independent 60-culture amplification rates on strain 4-11B and one 60-culture rate on strain 4-6D.

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Fluctuation Tests

We performed fluctuation tests on the 12 candidate HAR strains with thehighest frequency of amplifications. Fluctuation tests were adapted fromthe mutation rate protocols described in Paquin et al. [1984, 1986] andDorsey et al. [1992]. A single colony from the original candidate HARstrain was inoculated into YEP glycerol and grown overnight at 30°C toavoid preselection of antimycin A–resistant mutants. Cells were theninoculated into 300 ml of YEP glucose and separated into 10 independentcultures. Cultures were grown at 15°C for 5 days, then plated ontoantimycin A plates and incubated at 30°C for 5 days. Although theADH4:CUP1 amplifications from the HAR screen were isolated aftergrowth at 30°C, we chose to perform the fluctuation analysis at 15°C toallow comparison to our previous amplification rate studies. All antimycinA–resistant mutants were tested for a concomitant increase in copperresistance when the frequency of antimycin A–resistant mutants in aculture was less than 30. A random sample of 20 to 30 antimycin A–re-sistant mutants per independent culture, including all antimycin A–resis-tant growth phenotypes, were tested for increased copper resistance whenthe frequency of antimycin A–resistant mutants was above 30. Isolatesbearing amplifications of theADH4:CUP1locus were identified by South-ern blot analysis of chromosome preparations separated on pulsed-fieldgels for all antimycin A, copper-resistant mutants. Only about 3% ofindependent cultures from parental strains 4-11B and 4-6D display spon-taneousADH4:CUP1amplification. Thus, very few independent culturesshould contain cells with detectable amplification of theADH4:CUP1locus, if the high frequency of amplification in the colony screened was theresult of an amplification event early in the growth of a colony (a jackpotevent), whereas many independent cultures withADH4:CUP1amplifica-tions would indicate the strain has a high amplification rate. The proportionof independent cultures with a detectableADH4:CUP1amplification was50% or more in five of the 12 candidate strains tested.

Treatment with DNA-Damaging Agents

Yeast cells were treated with MMS (Aldrich Chemicals, Milwaukee,WI) at 0, 5, 7, and 10 mM or hydrogen peroxide (H2O2; Fluka Chemicals,

Ronkonkoma, NY) at 0.0, 0.02, 0.05, 0.10, 0.125, and 0.25% solutions bythe method described by Ramotar et al. [1991]. The source for 254-nmUV-irradiation was a UV Stratalinker 2400 crosslinker (Stratagene, LaJolla, CA). For a quick screen for UV sensitivity (see Fig. 2), approxi-mately 13 106 cells of the strain to be tested were picked from plates andresuspended in sterile water in a 96-well plate. Three serial 1:10 dilutionswere made and cells replica-plated to new plates. Replica-plated cells werethen exposed in the dark to 100 J/m2 UV and then incubated at 30°C for 3days in the dark to prevent photoreactivation. A60Co source at theUniversity of Cincinnati, College of Medicine, was used for exposure tog-irradiation. Yeast cells were exposed tog-irradiation at 0, 40, and 10 Gyin 96-well plates. Yeast strain CCP8A, which carries a deletion ofRAD9and is sensitive to all four DNA-damaging agents, was used as a positivecontrol.

Identification of Gene Responsible for UV Sensitivityof HAR Strain B9-40

TheLEU2 gene of HAR strain B9-40 was partially deleted usingLEU2gene blaster pNK85 [Alani et al., 1987] to allow the use of a wild-typeLEU2-CEN4-based yeast genomic library (ATCC 77162, Vienna, VA).

Fig. 1. Southern blot analysis of amplifications. Southern blot probedwith ADH4:CUP1sequences showing examples of pulsed-field migrationpatterns of three classes of amplifications. Lane 1 contains DNA fromstrain A4C, a diploid strain made by crossing HAR parental strains 4-11Band 4-6D [Dorsey et al., 1993]. Lanes 2–15 contain DNA from independentantimycin A, copper-resistant mutants that have amplifiedADH4:CUP1DNA. Lanes 2–5 contain DNA from four independent antimycin A–resis-tant, copper-resistant strains that contain class I amplification structures.Lanes 6–9 contain DNA from four independent antimycin A–resistant,copper-resistant strains that contain class II amplification structures. Lanes10–15 contain DNA from six independent antimycin A–resistant, copper-resistant strains that contain class III amplification structures. ChromosomeVII, the location of theADH4:CUP1 cassette in the parent strain isindicated.

Fig. 2. Characterization of the UV sensitivity of HAR strain B9-40.Freshly grown cells were resuspended in water, diluted in a 96-well plate,replica plated, and irradiated with either 0 or 100 J/m2 UV. All strainscontain theADH4:CUP1amplification detection genetic background. UVsensitivity of strain B9-40. Haploid strain 4-11B is the parental strain ofHAR strain B9-40. Haploid UV-sensitive control strain CCP8A carries arad9 deletion. Homozygous diploids are denoted by 2N. 411BxB940 is adiploid strain derived from a back-cross of B9-40 to strain 4-11B. Segre-gation of the UV sensitivity phenotype is shown by two tetrads from411BxB940 containing the four products (spores 1A–1D and 2A–2D) of ameiosis (spores 1A–1D and 2A–2D).

328 Peterson et al.

The genomic library was transformed into B9-40-Dleu2 cells by standardLiOAc techniques [Sherman, 1991]. Sets of greater than 1000 transfor-mants were pooled and plated to leucine minus medium, and UV-comple-menting clones were enriched by UV-irradiation at 100 J/m2. This UV doseresults in a 20-fold difference in survival between parental 4-11B cells andB9-40 cells (20 vs. 1%, respectively). Surviving transformants were pickedand resuspended in sterile water in a 96-well plate, and were then replica-plated to new leucine minus medium and UV-irradiated at 0, 100, 120, and140 J/m2. Plasmids from transformants displaying UV resistance similar toparental strain 4-11B were prepared and retransformed into fresh B9-40-Dleu2 cells, to confirm that the UV-resistant phenotype was the result ofthe plasmid. A total of 2601 transformants from 10 transformant poolswere screened after enrichment to identify two independent plasmids, p724and p931, that complemented the UV-sensitive phenotype of B9-402Dleu2 cells upon retransformation. The ends of the yeast genomicDNA insert in p724 were sequenced using Sanger dideoxy sequencing ona Perkin–Elmer model 373 or 377 automated sequencer (Foster City, CA)at the University of Cincinnati Molecular Biology Core. DNA sequences of200 base pairs (bp) at the each end of the yeast genomic DNA insert inclone p724 were used to search theSaccharomyces cerevisiaegenomedatabase [Goffeau et al., 1997; http://genome-www.stanford.edu/Saccha-romyces/]. This analysis showed that the DNA repair helicase geneRAD3was located on the plasmid insert. To verify that theRAD3gene on clonep724 was responsible for the restoration of wild-type UV resistance, a3.5-kbKpnI–XbaI fragment containing only the wild-typeRAD3ORF wassubcloned into centromere plasmid pRS416, transformed into B9-40, andthe transformed strain was shown to have wild-type UV resistance.

Sequencing and Cloning the rad3-I463K Allele

The RAD3PCR1 (59-cggggtacccccgatttgactacactttaagaag-39) andRAD3PCR2 (59-gctctagagcaaaagcgtatcattgca-39) primers (University ofCincinnati Molecular Biology Core) were used to generate a PCR productfrom strain B9-40 genomic DNA, which spanned therad3gene from 35 bpupstream of the open reading frame to 35 bp downstream of the openreading frame. The PCR1 and PCR2 primers were designed to haveKpnIand XbaI sites, respectively, for subsequent cloning of therad3-I463Kallele. The PCR product was purified from PCR primers with a WizardPCR kit (Promega, Madison, WI). Sequencing primers positioned every250 bp across theRAD3open reading frame were used for Sanger dideoxysequencing as described earlier. The thermally stable, high-fidelitypfuDNA polymerase (Stratagene) was used to generate a RAD3PCR1–RAD3PCR2 PCR product containing therad3-I463K allele from strainB9-40 genomic DNA and theRAD3 allele from strain 4-11B genomicDNA and subsequently cloned intoKpnI–XpaI polylinker sites of a yeastcentromere (pRS416) and yeast integrating (pRS406) plasmids (Strat-agene). The sequence of the mutant 463 codon was verified by sequencingthese clones.

RESULTS

High Amplification Rate Screen

We screened 4758 mutagenized haploid yeast strains asdescribed in Materials and Methods to identify the five highamplification rate (HAR) strains whose amplification ratesare shown in Table I.ADH4:CUP1 amplifications weredetected in only six of the 180 independent 4-11B and 4-6Dparental strain cultures, resulting in an amplification rateestimate of 16 1 3 10210 amplifications/cell/generation(Table I). Candidate HAR strain cultures were grown tosimilar or lower cell densities than those of the parental

cultures at the time of antimycin A selection. The highfrequency of antimycin A–resistant mutants made it neces-sary to analyze a random sample of 20 antimycin A–resis-tant mutants per culture (including all growth phenotypes)for increased copper resistance. The total number of anti-mycin A–resistant mutants analyzed, however, was similarin the parental and HAR candidate strains (Table I). Sinceonly a fraction of the antimycin A–resistant mutants werescreened the amplification rates of the candidate HARstrains are minimal estimates. The percentage of antimycinA, copper-resistant mutants with detectableADH4:CUP1amplification for each strain varied from 99% in strainB3-10 to 4% in strain B12-123. However, the number ofindependent cultures with detectableADH4:CUP1amplifi-cations was always higher in the candidate HAR strains.This translated into an increased amplification rate estimatefor all five HAR candidate strains, from 40- to 600-foldhigher than the 13 10210 amplifications per cell per gen-eration observed in the parental cultures.

ADH4:CUP1 Amplification Structures

Our lab has characterized three generalADH2 andADH4amplification structure types: chromosomal [Paquin et al.,1992], extrachromosomal linear palindromes [Dorsey et al.,1992, 1993], and extrachromosomal circles [Dorsey et al.,1993]. The structures of theADH4:CUP1 amplificationsdetected during determination of the HAR strain amplifica-tion rates have not been characterized in detail. However,the amplifications we observed displayed pulsed-field mi-gration patterns similar to those of the previously analyzedstructures. We therefore classified the amplifications basedon their pulsed-field migration patterns using the followingcriteria (for examples, see Fig. 1). Class I amplificationstructures migrate similarly to extrachromosomal linear pal-indrome amplifications: they migrate to a position below thesmallest yeast chromosome and theADH4:CUP1genes onthe amplification DNA are usually present in high copynumber relative to the single copy ofADH4:CUP1 onchromosomeVII (Fig. 1, lanes 2–5). Class II amplificationstructures migrate similarly to chromosomal amplifications:they migrate to positions similar to yeast chromosomes andthe ADH4:CUP1 genes on the amplification DNA arepresent in approximately equal copy number to the singlecopy of ADH4:CUP1 on chromosomeVII (Fig. 1, lanes6–9). Class III amplification structures migrate indepen-dently of pulse time, usually near the top of the pulsed-fieldgel, a characteristic of circular molecules [Hightower andSanti, 1989]. This class of amplifications also has a highcopy number ofADH4:CUP1 genes relative toADH4:CUP1 on chromosomeVII (Fig. 1, lanes 10–15). Finally,class IV amplification structures are multiple-amplificationstructures within a single antimycin A, copper-resistantcolony. None of the HAR strains gave rise to amplificationswith migration patterns characteristic of only a single class

Recombination and Gene Amplification 329

(Table II). Rather, all HAR strains gave rise to severalclasses ofADH4:CUP1amplifications.

UV Sensitivity of High Amplification RateStrain B9-40

We characterized the DNA damage sensitivity of theHAR strains because mutations in DNA repair genes havebeen shown to result in increased amplification rates inmammalian cells [Giulotto et al., 1991]. We found thatstrain B9-40 displays sensitivity to UV light (at a dose of100 J/m2, approximately 1% of strain B9-40 cells survivecompared to 20% of parental strain 4-11B cells; Fig. 2). Incontrast, B9-40 exhibited no increased sensitivity to MMS,H2O2, or gamma irradiation (data not shown). We detectedno increased sensitivity to these four DNA-damaging agentsin any of the other HAR strains. Genetic analysis suggestedthat both the UV sensitivity and the increased amplificationrate phenotypes of strain B9-40 were the result of a reces-sive mutation in a single gene (Fig. 2 and data not shown).

A Mutation in the RAD3 Gene Is Responsible for theUV Sensitivity of Strain B9-40

To identify the mutated gene in strain B9-40 responsiblefor enhanced gene amplification we made use of the coseg-regating UV-sensitivity phenotype. We screened a yeastgenomic library for clones that restored wild-type UV re-sistance in B9-40 cells. Two independent clones were iden-tified, p724 and p931, which restored wild-type UV resis-tance to B9-40 cells. The DNA sequences at each end of theyeast genomic DNA insert in clone p724 were used tosearch theSaccharomycesgenome database [Goffeau et al.,1997; http://genome-www.stanford.edu/Saccharomyces/].This analysis showed that the yeast genomic DNA in p724spanned base pairs 521,526 to 530,802 of chromosomeV.This region contains three full-length open reading frames(ORF): a hypothetical ORF (YER169W), an adenylate ki-nase gene (ADK2), and the DNA repair helicase geneRAD3. RAD3was the most likely gene to be responsible forthe rescue of the UV sensitivity of strain B9-40 since it hasbeen previously shown to be involved in nucleotide excisionrepair [Reynolds and Friedberg, 1981; Wilcox and Prakash,1981]. To verify that theRAD3 gene on clone p724 was

responsible for the restoration of wild-type UV resistance, aDNA fragment containing only the wild-typeRAD3 ORFwas subcloned into a centromere plasmid and shown torestore the UV resistance of strain B9-40. We confirmedthat the other clone that restores wild-type UV resistance toB9-40 cells, p931, also containedRAD3,by showing thatp931 has a DNA fragment that cross-hybridized to theRAD3gene of p724 (data not shown).

To identify the mutation responsible for the UV sensitiv-ity of strain B9-40, we sequenced therad3 gene from thisstrain. Only a single-base-pair difference from the publishedsequence at theSaccharomycesgenome database [Goffeauet al., 1997; http://genome-www.stanford.edu/Saccharomy-ces/] was detected, a T3 A transversion at base pair 1388.This predicts a change in the Rad3 protein at amino acid463, from an uncharged isoleucine (ATA) to a chargedlysine (AAA). A UV-sensitive mutation was previouslyidentified in this codon [Naumovski et al., 1985]. To dem-onstrate that AAA at codon 463 was not a polymorphismpresent in our strain background, we confirmed that thesequence of the parental strain 4-11B, from which B9-40was derived, was identical to the published sequence.

The rad3-I463K Allele Imparts an IncreasedAmplification Rate

To demonstrate that thisrad3-I463K allele was also re-sponsible for the high amplification rate of strain B9-40, wedetermined the amplification rates of isogenic strains whoseonly difference was an ATA or AAA at codon 463 ofRAD3.In strain B9-40 we replaced therad3-I463K allele with thewild-type RAD3allele and in strain 4-11B we replaced theRAD3 allele with rad3-I463K. The alleles of the isogenicstrains we constructed were confirmed by UV sensitivityand by sequencing across codon 463.

The amplification rates for the isogenic strains carrying awild-type RAD3allele in either the 4-11B or B9-40 strainbackground were similar to wild-type gene amplificationrates (Table III). In fact, only oneADH4:CUP1amplifica-tion was detected in the strains carrying a wild-typeRAD3gene. In contrast, strains carrying arad3-I463Kin either the4-11B or B9-40 background had substantially increasedrates of amplification. Indeed, 19 independent mutants car-rying amplifications were isolated from strains carrying therad3-I463Kallele. All three classes of amplifications weredetected among amplifications derived from HAR strainB9-40 and strains carrying therad3-I463Kallele. Thus, therad3-I463K allele imparts both a UV-sensitive phenotypeand an increased amplification rate.

Deletion of RAD52 Is Lethal in HAR Strain B9-40

We were interested to determine whether deletion ofRAD52 decreased amplification in the HAR strains. Al-though deletion ofRAD52 in the wild-type B3-10 and

TABLE II. Frequency of Classes of Amplification StructuresFrom HAR Strains

Strain Class I Class II Class III Class IV

4-11B 6/6 (100%) 0/0 (0%) 0/0 (0%) 0/0 (0%)B3-10 21/39 (54%) 2/39 (5%) 13/39 (33%) 3/39 (8%)B9-40 8/30 (27%) 11/30 (37%) 9/30 (30%) 2/30 (6%)B11-60 41/48 (85%) 0/48 (0%) 6/48 (13%) 1/48 (2%)B12-123 3/10 (30%) 5/10 (50%) 2/10 (20%) 0/10 (0%)B12-267 37/84 (44%) 10/84 (12%) 15/84 (18%) 22/84 (26%)

330 Peterson et al.

B11-60 backgrounds was straightforward, we were unableto obtain any yeast strains carrying deletions ofRAD52inthe B9-40 background. To determine whetherRAD52waslethal in this strain we transformed the B9-40 2N diploidstrain with theRAD52gene blaster and dissected tetrads. Ofthe 32 B9-40 2N tetrads we dissected, all four sporessurvived in 11 tetrads. Of the 28 B9-40 2NDrad52 tetradsdissected, no more than two spores survived in any tetradand only spores requiring uracil were recovered, indicatingthat RAD52 was not disrupted in any of the survivingspores. Thus, HAR strain B9-40 carries a mutation that islethal in aRAD52deletion background.

Amplifications Are Not Found in Strains WithDeletions of RAD52

Amplification rate estimates for HAR strains B3-10 andB11-60 and isogenic strains carrying deletions ofRAD52were performed simultaneously (Table IV). In every casewe were unable to detect amplifications in theRAD52-deleted strains, whereas amplifications were detected in allthe isogenic strains with wild-typeRAD52genes at similarrates to previous amplification rate estimates.

Deletion of MSH2 Increases Amplification Tenfold

The results from experiments showing that deletion ofRAD52decreased amplification in HAR strains suggestedthat recombination is indeed an important step in amplifi-cation. To confirm this we explored whether a mutationincreasing recombination would also increase amplification.Because the intramolecular recombination model predictsthat recombination between short similar DNA sequencesincreases amplification, we used strains with deletions of themismatch repair geneMSH2, which increases recombina-tion between DNA sequences with limited similarity [Selvaet al., 1995]. The mutations were constructed in the wild-type background as described in Materials and Methods.The MSH2-deletion strains had amplification rates approx-

imately 10-fold higher than those of the wild-type strains(Table IV). In addition, both class I and class III amplifi-cations were observed in theMSH2-deletion strains, sug-gesting that both linear extrachromosomal amplificationsand circular amplifications are increased by deletion ofMSH2.

DISCUSSION

We report here the use of theADH4:CUP1 system toidentify five strains ofS. cerevisiaethat display 40- to600-fold higher amplification rates than those of parentalstrains. These HAR strains are the first report of yeastamplificator strains. Although rare in normal cells, geneamplification is an important type of genomic rearrange-ment in carcinogenesis that can alter both chromosomalorganization and gene expression. The isolation of the HARstrains clearly demonstrates the utility of theADH4:CUP1system in studying gene amplification.

TABLE IV. Amplification Rates of HAR Strains CarryingDeletions ofRAD52 and MSH2

StrainNumber ofcells/culture

Number ofindependent

amplifications/total numberof culturesa

Amplification rateestimateb

B310 8.33 107 23/30 1.76 0.73 1028

B310,DRAD52 1.13 108 0/30 ,2.93 10210

B11-60c 1.23 107 13/60 26 1.13 1028

B11-60,DRAD52c 1.13 107 0/60 ,1.93 1029

411-B 5.53 107 0/30 ,6.23 10210

411-B,DMSH2 1.23 108 7/30 2.26 1.63 1029

aAmplifications determined by Southern blot analysis of chromosomesseparated on pulsed-field gels.bAmplification rate estimates and 95% confidence intervals calculated bythe P0 method of Lea and Coulson [1949].cData are a compilation of two independent 30-culture amplification rateestimation experiments.

TABLE III. Amplification Rates in Wild-Type and rad3-I463K Yeast Strains

StrainNumber ofcells/culture

Percentage of antimycinA, copper-resistant

mutants withADH4:CUP1 amplification

Number of independentamplifications/totalnumber of culturesa

Amplificationrate estimateb

4-11B/4-6Dc 2.93 108 60 (6/10) 6/180 16 1 3 10210

B9-40 1.63 108 18 (27/148) 22/50 46 2 3 1029

B9-40 RAD3 1.13 108 0 (0/21) 0/30 ,3 6 6 3 10210

B9-40 I463K 9.43 107 9 (14/150) 9/30 46 1 3 1029

4-11B RAD3 5.93 107 3 (1/34) 1/30 66 6 3 10210

4-11B I463K 6.93 107 17 (20/116) 14/30 96 2 3 1029

aAmplifications determined by Southern blot analysis of chromosomes separated on pulsed-field gels.bAmplification rate estimates and 95% confidence intervals calculated by the P0 method of Lea and Coulson [1949].cData are a compilation of two independent 60-culture amplification rate experiments using strain 4-11B and one 60-culture amplification rate experimentusing strain 4-6D.

Recombination and Gene Amplification 331

These HAR strains also provide new data about amplifi-cation mechanisms that are consistent with the intrachro-mosomal recombination model. First, identification of am-plifications in haploid strains at a rate similar to ourprevious estimates of amplification rates in diploids(;10210 amplifications per cell per generation) [Dorsey etal., 1992] suggests that the mechanism of amplification issimilar in both haploid and diploid cells. In addition, thepresence of both a normal chromosomeVII and an ampli-fication structure in haploid cells suggests that the amplifi-cation mechanism includes a replication step. Finally, noneof the HAR strains gives rise to just one type of amplifica-tion structure based on pulsed-field migration patterns.Therefore, the defect(s) in the HAR strains do not result inthe formation of any specific amplification structure. Rather,the rate of formation of both linear and circular extrachro-mosomal amplifications is increased in all HAR strains, aspredicted by the intramolecular recombination model.

The identification of a mutation in theRAD3 gene thatresults in a 40-fold increase in amplification rate representsthe first identification of a yeast gene whose product affectsthe amplification process. The Rad3 protein has an ATP-dependent DNA and DNA–RNA helicase activity with 59 to39 directionality [Sung et al., 1987a,b; Bailly et al., 1991];not only is it a component of the transcription and repairfactor TFIIH, but it also has an essential function in tran-scription [Feaver et al., 1993]. The known phenotypes ofRAD3 alleles suggest at least two possible roles for themutant Rad3-I463K protein in the amplification process thatfit well with proposed amplification models. One class ofrad3 mutations, therem alleles can increase both mutationand recombination rates and are lethal in aRAD52-deletionbackground [Hoekstra and Malone, 1984; Malone andHoekstra, 1984]. Both the lethality of theRAD52deletion instrain B9-40 and the moderate UV sensitivity of therad3-I463Kallele are consistent with the idea that therad3-I463Kallele is aremmutation [Montelone et al., 1988].RAD52isessential for double-strand break repair in yeast, so it hasbeen suggested that therem alleles ofRAD3 increase thefrequency of DNA double-strand breaks. DNA double-strand breaks have also been proposed as the initiating eventin amplification [Windle et al., 1991]. Thus therad3-I463Kallele could increase amplification simply by increasing therate of double-strand break formation.

An alternative role for therad3-I463Kallele is suggestedby the report of aRAD3allele that decreases the amount ofsimilarity required for recombination [Bailis et al., 1995;Maines et al., 1998]. Butler et al. [1995, 1996] propose thatrecombination between short inverted repeats is an essentialstep in gene amplification. Thus, there are at least twodifferent properties ofRAD3 that may affect amplificationformation. TheADH4:CUP1system can now be used to testspecific amplification models by examining the effects ofmutations and environmental factors that are predicted toaffect amplification. In addition, the identification of the

rad3-I463Kallele demonstrates that theADH4:CUP1sys-tem can be used to identify mutations that increase ampli-fication. Thus, theADH4:CUP1system is the first exampleof a eukaryotic system, in which mutations that affect am-plification can be identified and will therefore provide aunique tool for examining amplification mechanisms.

The functional human homolog [Sung et al., 1993] of theyeastRAD3 gene is thexeroderma pigmentosum group Dgene (XPD,also known asERCC2). Mutations in XP genesare responsible for the cancer-susceptibility syndrome, xe-roderma pigmentosum. Additionally, cells from XP patientshave been shown to display genomic instability, notablychromosome fragmentation and dicentric chromosomes[Casati et al., 1991; Huang et al., 1995] and a high level ofHa-ras oncogene amplification [Daya-Grosjean et al.,1993]. A predicted leucine to valine substitution in codon461 ofXPD, which aligns withRAD3codon 463, has beenpreviously identified as a mutation in an XP cell line [Weberet al., 1970; Frederick et al., 1994]. Thus, it would beinteresting to determine whether mutations in theXPDgeneincrease amplification in mammalian cells. The known ac-tivities of RAD3 in yeast suggest that mutations in otherhuman genes might also affect gene amplification.RAD3isa yeast DNA helicase involved in excision repair and tran-scription, and a mutation inERCC6,a mammalian helicasegene that is involved in the preferential repair of transcribedsequences, has been shown to increase amplification four-fold [Mondello et al., 1995]. Thus genes identified as af-fecting amplification in yeast should suggest good candi-dates for genes that affect amplification in mammalian cells.

The HAR strains have also allowed us to demonstrate thatRAD52 is essential for amplification of genomic DNA se-quences consistent with formation of these amplificationsby the intramolecular recombination model proposed byButler et al. [1996]. Previously, we could not test whetherany known gene was required in the amplification process,since amplification rates are so low in normal yeast cellsthat any decrease would be difficult to measure. The modestincrease in amplification rates in theMSH2-deletion strainfurther supports the concept that recombination betweenrepeats of limited similarity is an important step in ampli-fication. In addition, our data support an important role forrecombination in the formation of all three classes of am-plifications. In rDNA amplification inTetrahymena,recom-bination occurs between two identical 42-bp inverted re-peats. Such large, perfect inverted repeats are not found inthe yeast genome adjacent toADH4 [Goffeau et al., 1997;http://genome-www.stanford.edu/Saccharomyces/]. How-ever, several 8- to 10-bp inverted repeats are located be-tweenADH4and the centromere in a region where the noveljoints on several linear palindromes were mapped by re-striction analysis [Dorsey et al., 1992]. Recombination be-tween such repeats should be suppressed to low levels bythe cellular machinery that suppresses recombination be-tween similar but nonidentical sequences. This could par-

332 Peterson et al.

tially account for very low spontaneous amplification rates.Deletion of theMSH2 gene has been shown to increaserecombination approximately 10-fold between two similarbut not identical genes (75% identical with a maximum of15 matched and five mismatched continuous nucleotides ina stretch) but have no effect on homologous recombination[Selva et al., 1995]. Therefore, the increase in amplificationin the MSH2 deletion strain could be the result of anincrease in recombination between these short inverted re-peats.

ACKNOWLEDGMENTS

The authors thank J. Ma and K. Dixon for critical readingof the manuscript and K. Dixon, J. Strathern, and D. Gar-finkel for helpful discussions. C. Paquin is grateful forsabbatical support from the NCI–Fredrick Cancer Researchand Development Program.

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Accepted by—B. Kunz

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