High‐resolutiongenome‐wideanalysisofirradiated(UVandgammarays)diploid

yeastcellsrevealsahighfrequencyofgenomiclossofheterozygosity(LOH)events

JordanSt.Charles*,§,§§,EinatHazkani‐Covo*,§§,YiYin*,SabrinaL.Andersen*,FredS.

Dietrich*,PatriciaW.Greenwell*,EwaMalc**,PiotrMieczkowski**,ThomasD.Petes*

*DepartmentofMolecularGeneticsandMicrobiologyand§DepartmentofPharmacology

andCancerBiology,DukeUniversityMedicalCenter,Durham,NC27710

**DepartmentofGenetics,UniversityofNorthCarolinaatChapelHill,ChapelHill,NC

27599

§§contributedequallytothestudy

Genetics: Published Articles Ahead of Print, published on January 20, 2012 as 10.1534/genetics.111.137927

Copyright 2012.

2

Runninghead:High‐resolutiongenomicmappingofLOH

Keywords/phrases:mitoticrecombination,lossofheterozygosity,DNArepair,

Saccharomycescerevisiae,single‐nucleotidepolymorphism(SNP)microarrays.

Correspondingauthor:

ThomasD.Petes

DepartmentofMolecularGeneticsandMicrobiology

Box3054,DukeUniversityMedicalCenter

Durham,NC27710

Telephone:(919)684‐4986

Fax:(919)684‐6033

Email:tom.petes@duke.edu

3

ABSTRACT

Indiploideukaryotes,repairofdouble‐strandedDNAbreaks(DSBs)byhomologous

recombinationoftenleadstolossofheterozygosity(LOH).Mostpreviousstudiesofmitotic

recombinationinS.cerevisiaehavefocusedonasinglechromosomeorasingleregionof

onechromosomeatwhichLOHeventscanbeselected.Inthisstudy,weusedtwo

techniques(single‐nucleotidepolymorphism[SNP]microarraysandhigh‐throughputDNA

sequencing[HTS])toexaminegenome‐wideLOHinadiploidyeaststrainataresolution

averagingonekb.WeexaminedbothselectedLOHeventsonchromosomeVand

unselectedeventsthroughoutthegenomeinuntreatedcells,andcellstreatedwitheither

radiationorultravioletradiation(UV).Ouranalysisshows:1)spontaneousanddamage‐

inducedmitoticgeneconversiontractsaremorethanthreetimeslargerthanmeiotic

conversiontracts,andconversiontractsassociatedwithcrossoversareusuallylongerand

morecomplexthanthoseunassociatedwithcrossovers,2)mostofthecrossoversand

conversionsreflecttherepairoftwosisterchromatidsbrokenatthesameposition,and3)

bothUVandradiationefficientlyinduceLOHatdosesofradiationthatcauseno

significantlossofviability.UsingHTS,wealsodetectednewmutationsinducedbyγ‐rays

andUV.Toourknowledge,ourstudyrepresentsthefirsthigh‐resolutiongenome‐wide

analysisofDNAdamage‐inducedLOHeventsperformedinanyeukaryote.

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INTRODUCTION

AllorganismsexperienceDNAdamagefrombothexogenousandendogenoussources.

EndogenousDNAdamageincludesspontaneousdeaminationofnucleotides,

depurination/depyrimidination,oxidativedamage,anddouble‐strandedDNAbreaks

(DSBs)(FRIEDBERGetal.2006).DSBsarelikelytobeparticularlydeleterious,since

unrepairedDSBscanleadtochromosomerearrangementsorchromosomeloss.Although

thesourcesofendogenousDSBshavenotbeencompletelydetermined,someDSBsappear

toreflectnucleaseprocessingofsecondaryDNAstructures(suchasDNA“hairpins”)or

head‐oncollisionsbetweenthereplicationandtranscriptionmachineries(AGUILERA2002).

Below,inadditiontoexaminingspontaneousrecombinationeventsthatpresumablyreflect

therepairofendogenousDNAdamage,wealsoanalyzerecombinationeventsinducedby

twoexogenoussources:γraysandultravioletlight(UV).

BothγraysandUVcauseavarietyofdifferenttypesofDNAdamage.rayscauseDSBs,

single‐strandedDNAnicks,andbasedamage(FRIEDBERGetal.2006;WARD1990).UV

resultsinpyrimidinedimers(FRANKLINetal.1985;SETLOW1966),DNA‐DNAorDNA‐

proteincrosslinks(PEAKandPEAK1986),andsingle‐strandedDNAnicksresultingfromthe

dimerexcision(BREENandMURPHY1995).

Inyeast,asinmosteukaryotes,therearetworecombinationpathwaysthatareusedto

repairDSBs:non‐homologousend‐joining(NHEJ)andhomologousrecombination(HR).In

NHEJevents,asthenameimplies,brokenDNAmoleculesarere‐joinedbyamechanism

thatrequireslittleornohomology(DALEYetal.2005).Thismechanismismostactivein

haploidyeastcellsduringG1ofthecellcycle(SHRIVASTAVetal.2008).Indiploidcells,the

dominantpathwayisHR.HRusesanintacthomologousDNAmolecule,eitherthesister

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chromatidorthehomologouschromosome,asatemplateforrepairofthebroken

chromosome.

DSBscanberepairedbyseveraldifferentHRpathways(HEYERetal.2010).Therepair

ofaDSBbygeneconversionunassociatedwithacrossoverisshowninFig.1A.This

processinvolvesthenon‐reciprocaltransferofsequencesfromtheintactdonormolecule

tothebrokenchromosomeinseveralsteps:1)invasionofonebrokenendintotheintact

templatemolecule,followedbyDNAsynthesisprimedbytheinvading3’strand,2)removal

oftheinvadingendandreannealingofthisendbacktotheotherbrokenend,forminga

heteroduplexwithmismatches,and3)repairofthemismatches.Thismechanism

(synthesis‐dependentstrand‐annealingorSDSA)wasfirstsuggestedtoexplainsome

featuresofmeioticrecombinationinyeast(ALLERSandLICHTEN2001).Inthesecond

pathway(Fig.1B),geneconversionmaybeassociatedwithcrossovers.Inthispathway,a

doubleHollidayjunction(dHJ)isformedthatcanberesolvedtoyieldacrossoverornon‐

crossover.Inthispathway,heteroduplexesflanktheoriginalpositionoftheDSB.Although

theheteroduplexregionshavethesamesizeinFig.1B,inbothmeiosis(JESSOPetal.2005;

MERKERetal.2003)andmitosis(MITCHELetal.2010;TANGetal.2011),theconversion

tractsflankingtheDSBareoftenofdifferentlengths.ThedHJcanalsobedissolvedwithout

nucleolyticcleavageofDNAstrandstoyieldnon‐crossoverproductswithheteroduplexes

locatedincisononeofthetwointeractingchromosomes(HEYERetal.2010).Inthethird

pathway(Fig.1C),onepartofthebrokenDNAmoleculeislostandacompletechromosome

isthenreconstructedbybreak‐inducedreplication(BIR).Inthismechanism,oneofthe

brokenendsinvadestheintacttemplatemoleculeandareplicationforkissetupthat

duplicatesthetemplatefromthesiteofinvasiontothetelomere.

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IfHRinvolvesaninteractionbetweentwohomologuesthatcanbedistinguishedby

single‐nucleotidepolymorphisms(SNPs),conversionswithoutcrossoverswillproducea

smallpatchofLOHwithinachromosomethatisotherwiseheterozygous,whereasboth

crossoversandBIRresultinLOHthatextendsfromthesiteatwhichtheeventinitiatesto

theendofthechromosome.RepairofaDSBbyHRinwhichthehomologouschromosome

isusedasatemplatewillresultinLOH,butrepaireventsinvolvingthesister‐chromatid

willnot.Althoughmostsister‐chromatidrecombinationeventsaregeneticallysilent,

unequalsister‐chromatidexchangescanbedetectedinyeastbyavarietyofdifferent

systems(PETESandHILL1988).Usingoneofthesesystems,KADYKandHARTWELL(KADYK

andHARTWELL1992)showedthat,indiploidcells,sisterchromatidsarethepreferred

substratefortherepairofDSBsgeneratedbyX‐rays.Despitethispreference,itisclearthat

ionizingradiationandUVstronglystimulateHRevents(bothmitoticcrossoversandgene

conversions)betweenhomologouschromosomes(FABRE1978;NAKAIandMORTIMER1969).

Oneproblemwithstudyingspontaneousmitoticrecombinationisthatmostanalytic

systemsdonotallowtheselectionofbothdaughtercellsthatcontaintherecombinant

chromosomes.Severalyearsago,wedevelopedamethodofselectingreciprocalcrossovers

onchromosomeVthatsurmountsthisdifficulty(BARBERAandPETES2006;LEEetal.2009).

OnecopyofchromosomeVhasthecan1‐100allele(anochremutation)and,intheother

copy,theCAN1geneisreplacedbySUP4‐o,ageneencodinganochre‐suppressingtRNA

(Fig.2A).Intheabsenceofthesuppressor,strainswiththecan1‐100alleleareresistantto

canavanine,butbecauseofthesuppressor,thediploidusedinourexperimentsis

canavanine‐sensitive.Inaddition,thediploidishomozygousfortheade2‐1mutation(an

ochremutation).Strainswiththismutation,intheabsenceoftheSUP4‐ogeneformred

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colonies,butformpinkcoloniesifonecopyoftheSUP4‐ogeneispresent.Thus,thediploid

strainiscanavanine‐sensitiveandformspinkcolonies.Ifacrossoveroccursbetweenthe

centromereofchromosomeVandthecan1‐100/SUP4‐omarkers(adistanceofabout120

kb),acanavanine‐resistantred/whitecolonyisformed(Fig.2A).

Althoughthismethodwasfirstusedindiploidstrainslackingpolymorphisms,

subsequentstudiesweredoneinwhichadiploidwasconstructedusingtwohaploid

strainsthathadabout0.5%sequencedivergence(LEEetal.2009;LEEandPETES2010),

resultinginabout55,000SNPsdistributedthroughoutthegenome.Crossoversand

associatedgeneconversionsonchromosomeVweremappedtoaresolutionofabout4kb

byusingPCR,restrictiondigests,andgelelectrophoresistolookforLOH.Althoughafewof

thecrossovershadnoassociatedconversion,mostofthecrossoverswereassociatedwith

anadjacentconversionevent(boxedregionsinFig.2).Inthe3:1classofevents(Fig.2B),

intheboxedregion,threeofthefourchromosomeshadoneofthetwoformsoftheSNP

andoneofthechromosomeshadtheotherform(onesectorbeinghomozygousforaSNP

withtheothersectorbeingheterozygous).Inaddition,about40%ofthecrossoverswere

associatedwithaconversioneventinwhichthesameSNPwashomozygousinbothsectors

(Fig.2C);wetermtheseevents“4:0conversions.”Theobservationofthe4:0eventsargues

thatabouthalfofmitoticcrossoversresultfromtherepairoftwosisterchromatidsthatare

brokenatapproximatelythesamepositions.Onesimplemechanismforgeneratingthis

intermediateistohavetheDSBoccurinG1,andthebrokenchromosomereplicatetoform

twobrokenchromatids(LEEetal.2009).Thisproposedmechanismwasconfirmedby

analysisofthetypesofconversioneventsstimulatedbyγraysinsynchronizedG1andG2

cells(LEEandPETES2010).Inadditionto3:1and4:0conversionevents,3:1/4:0hybrid

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tractsarealsoobserved(LEEetal.2009;LEEandPETES2010).Suchtractscanalsobe

explainedasaconsequenceofrepairoftwobrokensisterchromatids(Fig.3).

Inthecurrentstudy,weuseSNPmicroarrays,andHTStomapselectedeventson

chromosomeVaswellasunselectedeventsthroughoutthegenomeataresolutionofabout

1kb.Toourknowledge,thesestudiesarethefirsttomeasurethenumbersandtypesof

LOHeventsthroughoutthegenomeinducedbydosesofionizingradiation(100Gy)andUV

(10‐15J/m2)thathavenosignificanteffectoncellviability.Wealsodeterminedthe

numberofmutationsinducedinthegenomesbythesetreatments.

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MATERIALSANDMETHODS

Strainsandgeneticmethods:Allexperimentswereconductedwiththediploidstrain

PG311(LEEetal.2009).TherelevantgenotypeofPG311isMATα::NATR/MATaURA3/ura3‐

1ade2‐1/ade2‐1TRP1/trp1‐1HIS3/his3‐11,15GAL2/gal2SUP4‐o/can1‐100

V9229/V9229::HYGV261553/V261553::LEU2.Thisdiploidwasgeneratedbycrossingthe

haploidstrainsPSL2andPSL5whichareisogenicwithstrainsW303aandYJM789,

respectively,exceptforalterationsintroducedbytransformation(LEEetal.2009).Below,

wewillrefertothehaploidparentsofPG311asW303aandYJM789.Ingeneral,PG311has

theSNPspredictedfromthehaploidparents.ThedisruptionofMATαinPG311allows

synchronizationofthisdiploidbyalphafactor.AlthoughdiploidsthatlackMATαdonot

sporulateundernormalconditions,suchstrainscanbesporulatedonplatescontaining5

mMnicotinamide(J.Rine,personalcommunication).Forexperimentsinwhichwe

analyzedmeioticproductsofPG311,thestainswerepre‐grownonYPDplateswith5mM

ofnicotinamide,andthentransferredtosporulationplatescontaining5mMnicotinamide.

Plateswereincubatedat25°for2‐4daysbeforetetraddissection.

Standardmediawereused(GUTHRIEandFINK1991)unlessnoted.Todetect

spontaneouscrossovers,wefirstisolatedsinglecoloniesofPG311grownonrichgrowth

medium(YPD)at30° fortwodays.Individualcoloniesweresuspendedin400μlofdH2O,

and100μlofthismixturewasplatedoncanavanine‐containingplates(SD‐arg+120μg/ml

canavanine).Theplateswereincubatedfourdaysatroomtemperature,followedby

incubationfor16hoursat4°;the4°incubationallowsbettervisualizationofthered

sectors.Wepurifiedcellsfromtheredandwhitesectorsforsubsequentanalysis.

10

Intheexperimentsinwhichcellswereirradiated,wesynchronizedcellsinG1using

alphafactor(LEEandPETES2010).Thesynchronizedcellsweretreatedwitheitherγ

radiationinaShepherdMark1Cesium‐137irradiatorat100GyorwithUV(254nm)

derivedfromaTL‐2000UltravioletTranslinkeratadosageof10or15J/m2.Following

radiation,thecellswereplatedeitheronnon‐selectiveplates(SD‐arg)orplatesthatlacked

arginineandcontained120μg/mlcanavanine.Thesubsequentgrowthofthecellsandthe

analysisofsectorsweredoneasdescribedaboveforthespontaneousselectionwiththe

exceptionthatsectoredcoloniesfortheUV‐treatedsampleswereisolatedfromnon‐

selectiveplatesgrownat30°insteadofroomtemperature.

SNPmicroarrays:designandoptimization:WedesignedtheSNParraysbasedon

genomicsequenceinformationavailablefromSGDforS288c(verycloselyrelatedto

W303a)(WINZELERetal.2003)andYJM789(WEIetal.2007).Microarraysthatwere

capableofdetectingLOHforSNPsinPG311weredesignedbasedonprinciplesoutlinedby

GRESHAMetal.(GRESHAMetal.2010).ForeachSNPrepresentedonthearray,four25‐base

oligonucleotideswereused:oneforeachstrandoftheW303aSNP,andoneforeachstrand

oftheYJM789SNP.TheSNPwaslocatedinthemiddleofthe25‐baseoligonucleotide.

Althoughthereareapproximately55,000SNPsinPG311,aboutthree‐quartersofthese

SNPswerenotusedforouranalysis.WeexcludedmostoftheSNPsfoundinrepeated

genes.Wealsoscreenedoutoligonucleotidesthathadameltingtemperatureforthe

perfectly‐matched25basepairduplexthatwaslessthan55°orgreaterthan59°.The

remainingoligonucleotides(representingabout15,000SNPs)thatwereprintedonthe

microarrayarelistedinTableS3.Wealsoincludedonthearrayabout120oligonucleotides

thatwerenotdifferentbetweenW303aandYJM789;thesearelistedinTableS4.

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OligonucleotideswereprintedontothemicroarraysbyAgilentTechnologiesinslides

containingabout105,000oligonucleotides.Manyoftheoligonucleotidesarerepresented

morethanonceinthemicroarrays.Followingexperimentstodeterminewhich

oligonucleotidesresultedinthemostspecifichybridizationsignals(describedin

SupportingInformation),wereducedthenumberofSNPsusedinouranalysisto13,000.

ThisfinalsetofoligonucleotidesispresentedinTableS5.

Methodsusedformicroarrayanalysis:samplepreparation,hybridization

conditions,anddataanalysis:Themethodsusedforsamplepreparation,hybridization

conditions,anddataanalysisaresimilartothosedescribedpreviously(LEMOINEetal.2005;

MCCULLEYandPETES2010).Adetaileddescriptionoftheseproceduresispresentedin

SupportingInformation.Inbrief,genomicDNAfromtheexperimentalstrainwaslabeled

withCy5‐dUTP,DNAfromthecontrolstrain(PG311)waslabeledwithCy3‐dUTP,andthe

twolabeledsampleswerecompetitivelyhybridizedtothemicroarrays.Thearrayswere

thenscannedatwavelengthsof635and532nmusingaGenePixscannerandGenePixPro

softwareusingsettingsrecommendedbythemanufacturer.Theratioofthemedians(635

nm/532nm;RM)foreachprobewasusedforanalysis,andreplicateRMswereaveraged.

ThedatawerecenteredaroundavalueofonebydividingeachprobeRMbytheaverageof

alloftheprobeRMsinordertonormalizefordifferencesinthehybridizationlevelsforthe

controlandexperimentalstrainsamples.

Wecalculated95%confidenceintervalsonthemediansizesofconversiontractsusing

TableB11ofAltman(ALTMAN1990).Comparisonsofconversiontractlengthsunder

differentexperimentalconditionsweredoneusingtheMann‐Whitneytestonthe

VassarStatsWebsite(http://faculty.vassar.edu/lowry/VassarStats.html).

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GenerationandanalysisofHTSdata:SampleswerepreparedforHTSasdescribed

abovefortheSNPmicroarraysamplepreparationwiththeexceptionthatgenomicDNA

wassonicatedto300‐700bpfragments.TheDNAwasthenpreparedforsequencingusing

theprotocolrecommendedbyIlluminafortheGenomeAnalyzerIIx.Thesampleswere

sequencedusinganIlluminaGAIIxmachine,generating67‐to75‐bppairedendreads.For

theeightsequencedsamples,coveragevariedfrom90‐to180‐fold.

ThedetailsoftheHTSdataanalysisarepresentedinSupportingInformation.Inbrief,

wedetectedregionsofLOHbyidentifyingSNPsintheexperimentalstrainsinwhichat

least90%ofthe“reads”thatwereoriginallyheterozygouswerenowidenticaltooneofthe

originalalleles.Weidentifiednewmutationsbyfindingbasesthatwereidenticalinthe

originaldiploid,buthadanovelbaseinatleast40%ofthe“reads”intheirradiateddiploid;

weusethe40%criterionbecauseweexpectthatanynewmutationwillbeheterozygous.

Mutationsthatappearedinmorethanoneindependentisolatewerenotcountedasdenovo

mutations,sincesuchmutationspresumablyaroseinthestrainbeforetreatmentwiththe

DNAdamagingagent.

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RESULTS

AsdescribedintheIntroduction,wepreviouslyselectedspontaneousmitotic

crossovers,aswellascrossoversinducedbyrays,thatoccurredbetweenthecentromere

ofchromosomeVandthecan1‐100/SUP4‐omarkers,anintervalofabout120kb(Fig.2)

(LEEetal.2009;LEEandPETES2010).Thediploidusedinthesestudies(PG311)was

constructedbyacrossbetweentwohaploidsthatareisogenicwithW303aandYJM789,

andisheterozygousforabout55,000SNPs.Inourpreviousanalysis,thepositionsofthe

crossoversandassociatedgeneconversioneventsweremappedtoaresolutionofabout4

kbusingaPCR‐basedstrategythatdeterminedwhethertheSNPswereheterozygousor

homozygous.Thisprocedureisimpracticalforgenome‐widemappingofrecombination

events.Below,wedescribetheuseofSNParraystomapspontaneous,UV‐inducedand

ray‐inducedcrossoversselectedonchromosomeV,aswellasunselectedcrossoversand

geneconversioneventsthroughoutthegenome.

SNParrayshavebeenusedpreviouslytomapLOHeventsintumorcells(LINDBLAD‐TOH

etal.2000),tomapmeioticrecombinationeventsinS.cerevisiae(MANCERAetal.2008),to

characterizechromosomerearrangementsandchromosomelossinC.albicans(ABBEYetal.

2011),andinavarietyofotherexperiments.UsingprinciplesoutlinedbyGRESHAMetal.

(GRESHAMetal.2010),wedevelopedaSNParraytoexamineLOHeventsthroughoutthe

genomeinthediploidPG311.Thisarrayhasoligonucleotidesthatdistinguishover13,000

SNPs,resultinginanaveragedensityofoneoligonucleotideperkbofgenomicDNA.The

detailsofthearraydesignandthespecificsequencesoftheprobesareinSupporting

MaterialsandMethodsandinTablesS3‐S5.

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Inourexperiments,welabeledgenomicDNAfromadiploidwitharecombination

eventwithonefluorescentnucleotideandDNAfromthecontroldiploidPG311witha

differentfluorescentnucleotide.Thesamplesweremixedandcompetitivelyhybridizedto

theSNParrays.IfSNPsretainedheterozygosityintheexperimentalstrain,thenthe

hybridizationsignalfortheoligonucleotidesrepresentingthatSNPwassimilartothatof

thecontrolstrain.LOHwasdetectedbyanincreaseinhybridizationtooligonucleotides

thathadoneformoftheSNP(forexample,theW303aform)andadecreasein

hybridizationtooligonucleotidesthathadtheotherform(forexample,theYJM789form).

Asdescribedabove,crossovereventsbetweenCEN5andthecan1‐100/SUP4‐o

markersproducecanavanine‐resistantred/whitesectoredcolonies.Thus,allofthe

samplesanalyzedhadaselectedrecombinationeventonchromosomeV,andwefoundthat

samplestreatedwithUVorγraysalsohadunselectedeventsonotherchromosomes.We

isolatedgenomicDNAfromboththeredandwhitesidesofthesectorsandexaminedthe

DNAbySNParraysfordiploidsuntreatedwithrecombinogenicagents,aswellasfor

diploidstreatedwithUVorγrays.AnexampleoftheanalysisforchromosomeVfora

selectedspontaneouscrossover(PG311‐2A)isshownonFig.4.InFig.4A,alow‐resolution

depictionofthehybridizationlevelsisshownforboththered(top)andwhite(bottom)

sectors.Inbothsectors,thehybridizationpatternindicatesthatthetransitionfrom

heterozygousSNPstohomozygousSNPsisatapproximatelySGDcoordinate55000.As

expected,theDNAthatiscentromere‐distaltothecrossoverjunctionfromtheredsector

hybridizeswelltotheW303a‐specificprobes(redline)andpoorlytotheYJM789‐specific

probes(blueline),sincetheredsectorisgeneratedbyLOHeventsthatincludethecan1‐

100markerthatisderivedfromtheW303a‐relatedhomologue(Fig.2).GenomicDNAfrom

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thewhitesectorshowsthereciprocalpatternofhybridization.The“spike”ofincreased

hybridizationintheredsectorforYJM789SNPsnearSGDcoordinate30000isanartifact

resultingfromadeletionofYJM789sequencesduringtheinsertionoftheSUP4‐ogeneinto

theYJM789‐derivedchromosome.

InFig.4B,weshowthesamecrossovereventathigherresolution.Eachsquareinthis

figureshowsthehybridizationratiotoaspecificoligonucleotide.Intheredsector,the

transitionbetweenthehomozygousSNPsandtheheterozygousSNPsisbetweenSGD

coordinates57170and60701.Inthewhitesector,thetransitionoccursbetween51915

and53692.Thus,thereisaregion(boxedinFig.4B)inwhichonesectorishomozygousfor

SNPsandtheotherisheterozygous.Thisregionisa3W:1Ygeneconversiontract(WandY

indicatingW303a‐derivedandYJM789‐derivedSNPs,respectively),equivalenttothe

boxedregioninFig.2B;inoursubsequentdiscussions,a3:1conversioneventindicates

3W:1Yconversionand1:3indicates1W:3Yconversion.Weestimatethelengthofthegene

conversiontractbyaveragingthemaximallengthofthetract(thedistancebetween

markersthatarenotwithinthegeneconversiontract,8.8kb)andtheminimallengthofthe

tract(thedistancebetweentheconvertedmarkers,3.5kb).ForthetractshowninFig.4B,

thislengthisabout6.2kb.Itisimportanttoemphasizethatthepresenceandextentof

geneconversiontractscanonlybeidentifiedwhenthepatternsofLOHareanalyzedin

genomicDNAfrombothsectorsofthesectoredcolony.

Withthegenome‐wideSNPanalysis,wefoundthattheparentaldiploidPG311andall

ofitssubsequentderivativeshadtwoLOHeventsthatwereunexpectedfromthesequence

oftheparentalhaploids.AllstrainswerehomozygousforW303a‐derivedSNPs

centromere‐distaltoSGDcoordinate685kbonchromosomeXIII,andwerehomozygous

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forW303a‐derivedSNPsbetweencoordinates412715and414085onchromosomeX.

Sincetheseevents,presumablygeneratedduringsub‐culturingofPG311,werepresentin

allstrains,theywereexcludedfromouranalysis.

AnalysisofspontaneousselectedLOHeventsonchromosomeVbySNParrays:

WeexaminedbySNParraysgenomicDNAfromboththeredandwhitesectorsfrom

fourteenindependentcanavanine‐resistantcolonies.Thecrossovereventsinfiveofthe

isolateshadbeenmappedpreviouslybyPCR‐amplifyingregionsalongtheleftarmof

chromosomeVthatcontainedpolymorphicrestrictionenzymesitesandtestingfor

homozygosityorheterozygositybyarestrictionenzymedigestanalysis(LEEetal.2009).

WealsomappedanadditionalnineeventssolelybySNParrays.Inthepreviousstudy,we

used34markersinthe120kbCEN5‐can1‐100/SUP4‐ointerval.Wemonitored172

markersinthissameintervalbySNParrays.

Inourpreviousstudy,wefoundthatmostofthespontaneouscrossoverswere

crossoverswithoutconversions(Fig.2A),crossoverswithassociated3:1conversions(Fig.

2B),crossoverswith4:0conversions(Fig.2C),orcrossoverswithhybridconversions

(3:1/4:0or3:1/4:0/3:1tracts;Fig.3)(LEEetal.2009).Acomparisonofthemappingof

recombinationeventsbythePCR‐basedmethodandSNParraysforfourofthesectored

coloniesisshowninTable1.Inthistable,wedefinethepositionofthecrossoverswith

onlytwoSGDcoordinates:thepositionofthecentromere‐proximalheterozygousmarker

thatisclosesttothecrossover/conversioneventandthepositionofthecentromere‐distal

homozygousmarkersrepresentingthecrossover.Althoughtheagreementbetweenthe

twomethodswasreasonablygood,asexpected,theSNParraymappedeventswithbetter

resolutionandalsorevealedthatsomeoftheconversioneventsweremorecomplexthan

17

previouslydetermined.Forexample,inPG311‐1.4,wepreviouslymappedacrossover

betweenSGDcoordinates125754and133080thatappearedtobeunassociatedwithgene

conversion.WiththeSNParrays,wemappedthetransitionathigherresolutionbetween

SGDcoordinates127038and130096,andwedetectedaSNPinthisregionthathad

undergonegeneconversion.Thecompletedescriptionofallspontaneouscrossoversand

associatedgeneconversioneventsisgiveninTableS6.

Oneexceptiontothegenerallygoodagreementbetweenthetwomappingmethodsis

isolatePG311‐1.6.Thiseventwasoriginallyclassifiedascrossoverassociatedwitha

conversiontractthatextendedfromSGDcoordinate31694to63936.SNPanalysis

demonstratedthatthewhitesectorhadaterminaldeletiononchromosomeV,beginning

nearcoordinate62000.Thesamesectoralsohadalargeterminalduplicationon

chromosomeVII.Althoughthisrearrangementhasnotbeenfullycharacterized,sincethere

areaclusterofdeltaelementsnearthebreakpointonchromosomeVandTyelementsat

thebreakpointonchromosomeVII,itispossiblethatthestrainhasachromosomeV‐VII

translocation,similartothosethatwehavecharacterizedpreviously(ARGUESOetal.,2008).

NoalterationsweredetectedoneitherchromosomeVorVIIintheredsector.Sequence

analysisindicatedthattheredsectorretainedtheSUP4‐ogene.Itispossiblethatthecell

thatgaverisetotheredsectorlosttheprionPSI,thataffectstheefficiencyofochre

suppressors(SHKUNDINAandTER‐AVANESYAN2007),althoughotherpossibilitiescannotbe

excluded.WhateverthedetailsofthegeneticalterationsinPG311‐1.6,theeventdoesnot

representaconventionalalleliccrossoveronchromosomeVand,therefore,isexcluded

fromouranalysis.

18

Ofthe13colonieswithspontaneousreciprocalrecombinationeventsanalyzedbySNP

arrays,thenumbersofcoloniesofvariousclasseswere:1)twocrossoverswithout

detectableconversions,2)twocrossoverswith3:1conversionevents,3)onecrossover

witha0:4conversion,4)onecrossoverassociatedwithahybridtract(1:3and0:4

segments),and5)sevencrossoverswithcomplexconversiontracts(Table2).Thecomplex

conversiontractswillbediscussedfurtherbelow.

Thelocationsofthespontaneouscrossoversandassociatedconversiontractsare

showninFig.5A.Eachsectoredcolonyisdepictedasapairoflineswiththeupperline

representingtheredsectorandthelowerlinerepresentingthewhitesector.Theredand

blacklinesegmentsindicatethatthesectorishomozygousfortheW303a‐associatedSNPs

andtheYJM789‐relatedSNPs,respectively.Thegreenlinesegmentindicatesthatthe

sectorisheterozygousfortheSNPs.Weshowthetwochromosomeswithineachsectorasa

singlelinebecauseouranalysisdoesnotallowustodeterminethecouplingrelationships

forheterozygousSNPsbetweenthetwohomologues.Themedianlengthofallcrossover‐

associatedconversiontractswas6.1kb,similartothemedianobservedinourprevious

studyofspontaneousconversiontracts(6.5kb,(LEEetal.2009).OnlyoneunselectedLOH

wasobservedinunirradiatedcells.BothsectorsinPG311‐7Bhadageneconversionevent

onchromosomeVIIIunassociatedwithacrossover(TableS6).Thus,thefrequencyof

spontaneousunselectedLOHevents/cellisverylow(about0.08)asexpected.

FortheeventsshowninFig.5A,3:1conversioneventscouldreflectaninitiatingDNA

lesionoccurringanywherewithinthetract,sinceeventscanbepropagatedbidirectionally

fromtheDSB(TANGetal.2011).For4:0or3:1/4:0hybridtracts,theinitiatinglesion

presumablyoccurswithinthe4:0regionofthetract(Fig.3).Althoughwedonotseeany

19

stronghotspotsforspontaneouseventswiththislimiteddataset,inalargersample,we

foundthattheregionbetweenSGDcoordinates41,000and60,000hadasignificantly

elevatedlevelofcrossoversandtheregionnearCEN5hadasignificantlyreducedlevelof

events(LEEetal.2009).

AsshowninFig.5A,manyoftherecombinationeventsareassociatedwithmultiple

transitionsbetweenheterozygosityandhomozygosity.InTableS6,foreachsectored

colony,weassignedalettertorepresenteachtransitionpoint;foreachtransitionpoint,we

alsoshowtwoSGDcoordinates,indicatingthepositionsoftheclosestSNPsonthearray

thatflankthetransitionpoints.Thesimplestevents(crossoverswithoutgeneconversion,

6Band1.7inFig.5A)haveasingletransitionpoint.TherecombinationeventshowninFig.

4(whichcorrespondstoevent2AinFig.5A)hastwotransitionpointsatdifferent

positions,oneintheredsectorandoneinthewhitesector.Incontrasttotheserelatively

simpleevents,thesectoredcolony18A(Fig.5A)hassixtransitions,oneintheredsector

andfiveinthewhitesector.Inouranalysis,ifthetransitionpointisidenticalinboth

sectors,itisonlycountedonce.InTableS6,wealsoassignaclass(AtoL)forallevents.In

TablesS1andS2,eachclassofeventisdiagrammedusingthesameapproachemployedin

Fig.5A.Inthesefigures,wealsoindicatethenumberofeventsineachclass,andwhich

supplementaryfigure(Figs.S1‐S40)showsthepatternofDNArepaireventsconsistent

withthespecificconversionevent.Thesamemethodsareusedtodescribethe

recombinationeventsinducedbyDNAdamageaswereusedtodescribethespontaneous

events.Multipletransitionswithinconversiontractscouldreflect“patchy”repairof

mismatcheswithinlongheteroduplexes(discussedfurtherbelow)ortemplateswitching

20

betweenhomologues.ForthespontaneousconversioneventsinFig.5A,wedidnotfinda

correlationbetweenSNPdensityandthenumberoftransitionswithinthetract(r2=0).

AnalysisofLOHeventsbySNParraysincellstreatedwithγrays:Weanalyzed

PG311sectorsthatweregeneratedinapreviousstudy(LEEandPETES2010)bytreatment

ofG1‐arrestedcellswith100Gyofγradiation,followedbyselectionofred/whitesectored

coloniesoncanavanine‐containingplates;thisdoseofγrayselevatedthefrequencyof

sectoringabout26‐fold.AllofthecoloniesexaminedhadacrossoveronchromosomeV.

WeanalyzedsevenofthesesectoredcolonieswithSNPmicroarrays,andtwoofthesewere

alsoexaminedbyHTS.TheSNParraydataareshowninTableS7withdepictionofthe

recombinationeventsinTablesS1andS2.

Thepositionsoftheselectedcrossoversandassociatedgeneconversioneventsinthe

CEN5‐can1‐100/SUP4‐ointervalareshowninFig.5B.OurmappingoftheseeventsbySNP

arraysisinreasonablygoodagreementwithourPCR‐basedmappingmethod(LEEand

PETES2010).AlloftheconversioneventshadatleastoneSNPthatwashomozygouson

bothsidesofthesector(4:0conversion)asexpectediftherecombinationeventswerea

consequenceofrepairoftwosisterchromatidsbrokenatthesameposition(Figs.2and3).

Inadditiontotheselectedevents,fromourgenome‐wideanalysis,weobserved17

unselectedeventsonotherchromosomesamongthesevencolonies:fourcrossovers

associatedwithconversion(Table2),elevenconversionsthatwerenotassociatedwith

crossovers,andtwobreak‐inducedreplication(BIR)events(Table3).Sincethefrequency

ofunselectedcrossoversinunirradiatedsamplesisverylow(lessthan0.1/cell),itislikely

thatalloftheeventsintheirradiatedcellsreflecttherepairofγray‐inducedDNAdamage.

ThelocationsoftheseunselectedeventsareshownasbluesymbolsinFig.6.Theevents

21

appearrandomlydistributedinthissmalldataset.TheSNParraysforradiation‐induced

unselectedcrossoversandassociatedconversionshavepatternssimilartotheselected

crossovershowninFig.4.Inaddition,weobservedmanyconversioneventsunassociated

withcrossovers(Fig.7).Thisfigureshowsatlowandhighresolutiona0:4conversion

eventinwhichbothsectorshavegainedYJM789SNPsandlostW303aSNPs.Thispattern

couldrepresentaneventthatoccurredpriortoradiation.However,sincesucheventswere

observedcommonlyinirradiatedcellsbutnotincontroldiploids,weassumethatmost(or

all)wereinducedbyγrays.

Sincetheredandwhitesectorsareproducedbythetwodaughtercellsresultingfrom

thedivisionofradiationtreatmentofaG1‐synchronizedcell,theanalysisofgenomicDNA

frombothsectorsgivesvaluablemechanisticinformationevenforunselectedevents.For

example,ifweobservedaninterstitialLOHeventbyexaminingonlythewhitesector,we

wouldnotknowwhetherthiseventwasaconsequenceofa3:1conversion,a4:0

conversion,oratwo‐stranddoublecrossover.Thisambiguitycanberesolvedby

examininggenomicDNAfromtheredsector.

WeobservedtwosectoredcoloniesthathadBIRevents.InsingleBIRevents(suchas

ClassL2inTableS1),onesectorhasanLOHeventthatextendsfromaninternalsiteonthe

chromosometothetelomere,whereastheothersectorisheterozygousforthesameSNPs.

IndoubleBIRevents,bothsectorshaveLOHeventsextendingfromaninternalsitetothe

telomere(ClassL1inTableS1).Interestingly,inthecolonywiththesingleBIRevent,there

isaconversioneventonthechromosomethatwasoriginallythesisterchromatidofthe

oneinvolvedinBIR.ThisresultarguesthatbothsisterchromatidshadDSBsat

22

approximatelythesameposition.ThemolecularinteractionsrequiredtoproduceClasses

L1andL2areshowninFigs.S39andS40.

Alloftheselectedandunselectedreciprocalcrossoversinducedbyγrayswere

associatedwithconversiontracts.Themedianlengthofallconversiontracts(both

associatedandunassociatedwithcrossover)was12.9kb(95%confidencelimitsof5.2‐

20.4kb).Themedianlengthsofconversiontractsassociatedandunassociatedwith

crossoverswere18.4kb(10.8‐25.3)and8.4kb(2.6‐13.3),respectively.BytheMann‐

Whitneytest,themedianlengthsofcrossover‐associatedandcrossover‐unassociated

ray‐inducedconversiontractsweresignificantlydifferent(p=0.01).

AnalysisofLOHeventsbySNParraysincellstreatedwithUV:G1‐synchronized

PG311cellsweretreatedwithaUVdoseof10‐15J/m2.Thisdoseresultedinnosignificant

lossofviabilitybutstimulatedthefrequencyofsectorsbyabout1000‐fold.Weexamined

threesectoredcoloniesbySNParraysandtwoofthesecolonieswerealsoanalyzedbyHTS.

InadditiontotheselectedcrossoveronchromosomeV,amongthethreecolonies,there

weresevenunselectedcrossovers,33unselectedconversionevents,andoneBIRevent

(Tables2and3).Thus,therewereaboutfourteenunselectedLOHeventsperUV‐treated

cell.ThelocationsofselectedchromosomeVeventsandtheunselectedLOHeventsare

showninFigs.5and6,respectively.TheUV‐inducedLOHeventsaredistributedfairly

evenlythroughoutthegenome(Fig.6).Asobservedfortheγray‐inducedBIRevents,the

UV‐inducedBIReventislocatedclosetothetelomere.Thedetailedinformationabout

breakpointsinUV‐treatedcellsisshowninTableS9andTablesS1andS2.

Allofthecrossovereventshadanassociatedconversion.Inmostoftheconversion

events,therewasatleastoneSNPwiththe4:0or0:4pattern,suggestingthatUV‐induced

23

damageinG1mayresultinDSBs(Fig.2B).ThemedianlengthofallUV‐inducedconversion

tractswas9.2kb(6.5‐10.3),whereasthemedianlengthsofconversiontractsassociated

andunassociatedwithcrossoverswere10.3(7.0‐18.9)and7.5(4.5‐10.2)kb,respectively.

Althoughthecrossover‐associatedconversionsarelongerthanthecrossover‐unassociated

conversions,thisdifferenceisnotsignificant(p=0.08byMann‐Whitneytest).

HTSanalysisofLOHinγray‐andUV‐treatedcells:SinceG1‐arrestedyeastcells

treatedwith100Gyofionizingradiationhaveabout35DSBspercell(LEEandPETES2010),

theaveragenumberofunselectedLOHeventspercell(twotothree)indicatesmostevents

mustberepairedbymechanismsthatdonotgenerateLOH.Analternativepossibilityis

thatasubstantialfractionoftheeventshaveshortconversiontractsthatarenotdetectable

bytheSNParrays.SinceHTScandetectLOHeventsforallofthe55,000SNPsexistingin

thediploidstrain,ratherthanthe13,000SNPsrepresentedontheSNParray,we

sequencedgenomicDNAsamplesfrombothredandwhitesectorsoftwosectoredcolonies

ofray‐treatedsamples(PG311‐GR‐37R/WandPG311‐GR‐40R/W)andtwocoloniesof

UV‐treatedsamples(PG311‐UV‐8R/WandPG311‐UV‐9R/W).Thedetailsofthisanalysis

aredescribedinSupportingInformation.AlloftheLOHeventsdetectedbySNParrays

werealsofoundbyHTS.LOHeventsthathadnotbeenpreviouslydetectedbytheSNP

arrayswereconfirmedbyre‐sequencingtherelevantPCRfragment.ThepatternsofLOHas

identifiedbyHTSintheγray‐andUV‐treatedsamplesareinTablesS8andS10.

Figure8showsacomparisonbetweenSNPmicroarrayandHTSdataforanLOHevent

onchromosomeIIinaUV‐treatedsample(PG311‐UV‐8R).TheSNPmicroarrayindicates

thatatransitionbetweenheterozygousandhomozygousSNPsoccurssomewherebetween

SGDcoordinates450919and452926,whereastheHTSdatarefinethemappingofthis

24

transitionbetweenSGDcoordinates451337and451581.Additionally,theHTSdata

showedthattherecombinationeventwasmorecomplexthanindicatedbythemicroarray

data.ByHTS,wefoundthattheregionbetweencoordinates448628and450279had

undergoneLOH;thiseventwasnotdetectedbymicroarraysbecausetherewereno

oligonucleotidesbetween448488and450919onthemicroarray.Asummaryofthe

comparisonofdatafromSNParraysandHTSforthesamesamplesisgiveninTable4.

AlthoughmoreLOH eventswereobservedwithHTSthanwithSNParrays,the

differencewasnotlarge.Forexample,inthetwoUV‐treatedsamples,weobserved32LOH

eventsbySNParraysand35eventsbysequencing.Intheγray‐treatedsamples,wefound

fiveeventsbySNParraysandsixeventsbysequencing.Sincemorethan80%oftheevents

detectedbyHTSwerealsodetectedbymicroarrays,itisunlikelythatourestimatesofLOH

eventsaresubstantiallyaffectedbyahighfrequencyofgeneconversioneventswithshort

conversiontracts.Wecannotruleout,however,thepossibilityofgeneconversionevents

withveryshort(<100bp)tracts.AsshowninTable4,anumberofthegeneconversion

tractsanalyzedbyHTSweremorecomplexthanthesametractsexaminedbytheSNP

arrays.Thefrequenciesofcomplextracts,asdeterminedbyHTSandSNPmicroarrays,

were0.37and0.22,respectively.DespitethedifferencesinthenumbersandtypesofLOH

eventsdetectedbyHTSandtheSNPmicroarrays,itisclearthatmostoftheLOHeventsare

detectablebytheSNPmicroarrays.

HTSanalysisofmutationsinducedinγray‐andUV‐treatedcells:About99.5%

ofthebasesbetweenthetwohomologuesofPG311areidentical.TheHTSdatagenerated

forthesamecoloniesexaminedforLOHeventswereanalyzedforradiation‐induced

mutations.WeanalyzedbothsectorsoftwosectoredcoloniesinducedbyUVandtwo

25

sectoredcoloniesinducedbyraysfornewmutations(TableS11).Therewerethreeand

twelvedenovopointmutationsdetectedintheray‐andUV‐treatedsamples,respectively.

Allthreeofthemutationsinducedbyγraysandsixofthetwelvemutationsinducedby

UVwereinbothredandwhitesectorsofthecolony.Thepresenceofthemutationinboth

sectorsindicatesthatthemutationinducedinG1bytheradiationwasrepresentedinboth

strandsoftheduplexpriortoreplication.ThemutationsintheUV‐treatedcellsthatwere

presentinonlyonesectorcouldreflectamutantbaseinonlyoneofthetwostrands.These

twotypesofeventshavebeenobservedpreviouslyinUV‐treatedcells(ECKARDTandHAYNES

1977;JAMESandKILBEY1977).NineoftwelveoftheUV‐inducedmutationsandtwoofthree

oftheγray‐inducedmutationsweretransitions.Inmuchmoreextensivestudyof

spontaneousandUV‐inducedmutationsattheSUP4‐olocus(KUNZetal.1987),

spontaneousmutationshadaratiooftransitions:transversionsof4:6,whereasUV‐induced

mutationswerebiasedtowardtransitions(4:1).

Most(11of15)oftheinducedpointmutationswerelocatedwithingenesratherthan

betweengenes(TableS11).ByChi‐squareanalysis,thedistributionofmutations

throughoutthegenomeisnon‐random(p=0.002).Fiveofthefifteenmutationsarelocated

ontheleftarmofXIand,remarkably,twomutations(oneinducedbyγraysandone

inducedbyUV)arewithintheNUP120gene.Bysimilarmethodsusedtodetectnewbase

substitutions,wefailedtodetectanyinsertion/deletionmutations(in/dels)intheeight

sequencedsamples.Itshouldbepointedout,however,thatdetectionofin/delsinHTSdata

withshort‐pairedreadsischallenging,particularlyinadiploidthatisheterozygousfor

manypre‐existingin/dels.

26

DISCUSSION

Inthisstudy,wemappedbothselectedandunselectedmitoticrecombination

eventsinagenome‐wideanalysis.MostoftheeventsweremappedusingonlySNP

microarrays,butfoureventswereexaminedbybothSNPmicroarraysandHTS.Toour

knowledge,thisstudyisthefirsttoexaminespontaneousandDNAdamage‐inducedLOH

eventsthroughouttheyeastgenome.Theconclusionsfromthisstudyare:1)thegene

conversiontractsanalyzedbySNParraysandHTSwereoftenmorecomplexthaninferred

fromourearlierlower‐resolutionmappingstudies(LEEetal.2009;LEEandPETES2010),2)

dosesofradiationthatresultinlittleornolossofcellviabilityinG1‐synchronizeddiploid

cellsresultedinmultipleunselectedLOHevents,and3)thesamedosesofγraysandUV

usedintheLOHstudyresultinverylowlevelsofdenovomutations.Inaddition,we

concludethat,althoughHTShasfour‐foldbetterresolutionthanSNPmicroarrays,theSNP

arraysdetectmostofthesameeventsidentifiedbyHTS.Theseconclusionswillbefurther

discussedbelow.

Lengthsofgeneconversiontracts:Themedianlengths(95%confidencelimits

showninparentheses),asmeasuredbySNParrays,ofgeneconversiontractsassociated

withcrossoversforspontaneous,UV‐induced,andray‐inducedeventswere6.1,10.3,and

18.5kb,respectively.Aswehaveobservedpreviously(LEEetal.2009),mitoticgene

conversiontractsaresubstantiallylongerthanmeioticconversiontracts(MANCERAetal.

2008).Inaddition,fortheray‐inducedconversionevents,theconversiontracts

associatedwithcrossoversweresignificantlylongerthanthoseunassociatedwith

crossoversashasbeenobservedpreviously(AGUILERAandKLEIN1989;MANCERAetal.

2008).Onesimpleexplanationofthisobservationisthatconversioneventsunassociated

27

withcrossoversusuallyareaconsequenceofSDSA,andsucheventsmightinvolvelimited

processingofthebrokenchromosomeends.Incontrast,crossoverslikelyproceedthrough

formationofadoubleHolidayjunction.FormationofthisdHJintermediatemayrequire

moreextensiveprocessingofbrokenDNAendsand/ormoreextensiveDNAsynthesis

primedfromtheinvadingend.ItisalsopossiblethatbranchmigrationofthedHJ

intermediatecouldextendthelengthoftheheteroduplexassociatedwiththecrossover;

thispossibilitywillbefurtherdiscussedbelow.

RecombinogenicDNAdamage:Althoughitisclearfromavarietyofstudiesthat

DSBsstimulatemitoticrecombination,theDNAlesionresponsibleforinitiating

spontaneousrecombinationeventsisnotcertain.Wepreviouslyshowedthatabouthalfof

mitoticcrossoversonchromosomeVareassociatedwithageneconversiontractsthatare

exclusively4:0or0:4orthathavearegionof4:0or0:4(hybridtracts).Suchconversion

tractsindicatethatbothsisterchromatidshavebreaksatapproximatelythesameposition

andonesimplemechanismconsistentwiththispropertyisthatthesespontaneous

conversioneventsreflectaDSBformedinG1ofthecellcycle.Supportingthisconclusion,

many(44%)oftheconversioneventsinducedbyγraysinG1ofthecellcyclehaveregions

of4:0or0:4segregation,whereasnoneoftheconversioneventsinducedbyγraysinG2

hadthispattern(LEEandPETES2010).Amongthemechanismsthatcouldproducethe

spontaneouslesionsrequiredtoinitiaterecombinationare:1)closely‐opposednicks

generatedduringremovalofadductscausedbyoxidativeDNAdamage,2)DSBscausedby

Top2porothercellularendonucleases,3)lesionsresultingfromcollisionsbetween

replicationforksandthetranscriptionmachinery,4)DSBsresultingfromthecollisionof

convergingreplicationforks,and5)nuclease‐dependentprocessingofsecondaryDNA

28

structures(“hairpins”andcruciforms).Thusfar,wehavebeenunabletoassociatethe

spontaneousrecombinationeventswithanyofthesemechanisms.Forexample,thetwo

positionsthatrepresenttheconvergenceofreplicationforksontheleftarmof

chromosomeV(FACHINETTIetal.2010)arenothotspotsforrecombinationinourlimited

dataset.IfthereareseveraldifferentmechanismsthatcanproducerecombinogenicDNA

lesions,wewillneedtomapmanyeventstodetectsignificantassociations.

Inourcurrentanalysisofγray‐inducedgeneconversioneventsinG1‐synchronized

cellsbySNPmicroarrays,wefoundthattenoftheelevenconversiontractsassociatedwith

crossovershada4:0or0:4segment,andeightoftheelevenconversiontractsthatwere

unassociatedwithcrossovershadsuchasegment.Thisobservationisconsistentwiththe

possibilitythatmostoftheobservedrecombinationeventsinIR‐treatedcellsreflecteda

DSBintroducedbyγraysinG1.

TherecombinogeniceffectofUV‐inducedDNAdamageislessclear.Onepossibilityis

thatsmallgapsresultingfromtheremovalofUV‐induceddimersaretherecombinogenic

lesion.GalliandSchiestl(GALLIandSCHIESTL1999)foundthatUVdidnotstimulatemitotic

recombinationbetweendirectrepeats(single‐strandannealing)inG1‐arrestedcellsunless

thecellswereallowedtoentertheS‐periodaftertheUVtreatment.Incontrast,whenG1‐

arrestedcellsweretreatedwithIR,stimulationofsingle‐strandannealingwasobserved

withoutrequiringthecellstoenterS.IftherecombinogenicDNAlesionisaDSB,thelikely

explanationofthedifferentresultsisthatIRdirectlycreatesDSBswhereastherepairofUV

lesionsresultsinnicksthatresultinDSBswhenthenickedmoleculeisreplicated(GALLI

andSCHIESTL1999).Bythisexplanation,itissurprisingthatmanyoftheconversionevents

inducedbyUVinG1inourexperimentshad4:0or0:4segments,suggestingthatthese

29

exchangeswereaconsequenceofaG1‐stimulatedDSB.SuchaDSBcouldbegeneratedin

G1iftheremovalofdimersonoppositeDNAstrandsresultedinaveryshort(<10bp)

duplexregionseparatingthe30bpgaps.Basedonpreviousestimatesofthenumberof

dimersinducedby20J/m2ofUV(DAIGAKUetal.2010),wecalculatethatthereareabout

7500dimers/diploidgenomeinducedbyaUVdoseof15J/m2and,basedonaPoisson

distribution,therewouldbeabout35regionspergenomeinwhichtwodimersareon

oppositestrandswithin75basepairsofeachother.SincetheDSBwouldrequiretwo

closely‐opposedgapsratherthantwoclosely‐opposeddimers,thekineticsofgap

formationandgaprepairaffecttheprobabilityofDSBformationbythismechanism.

Anothercomplicatingfactoristhatthefrequencyofclosely‐opposeddimersishigherthan

expectedifdimerformationisrandom(REYNOLDS1987).

AsecondpossibilityisthatDSBformationisinitiatedbygapsonoppositestrandsthat

arerelativelyclosetogether(<500bpapart),buttoofaraparttogenerateaG1DSB.Ifa

DNAmoleculewithsuchgapsisreplicated,theproductwouldbetwosisterchromatids

withDSBslocated500bporlessapart.Processingofthebrokenendstoyieldsingle‐

strandedDNAregions500bporgreaterwouldprecludeformationofadHJinvolvingthe

twosisterchromatids.Thus,suchmoleculeswouldlikelyberepairedusingtheintact

homologueasthetemplate,generatinga4:0conversion.Thismodelisconsistentwiththe

GALLIandSCHIESTLinterpretation.Wecalculatethatcellsirradiatedwith15J/m2would

haveabout234dimersonoppositestrandswithin500bpofeachother.

AnalternativepossibilityisthatrecombinationeventsareaconsequenceofDSBs

occurringatreplicationforksstalledatunexcisedpyrimidinedimers.UnrepairedUV‐

induceddamagehasbeendemonstratedtoblockreplicationforksandreplicationofsuch

30

damagedtemplatespromotessister‐chromatidrecombination(BRANZEIandFOIANI2010).

Althoughwecannotexcludethismodel,theobservedUV‐induced4:0eventswouldrequire

thatthereplicationforkstalledatthedimerresultintwobrokensisterchromatids,

perhapsbyincreasingtheprobabilityofareplicationforkcollision.Itshouldbe

emphasizedthatalthoughUVveryefficientlystimulatescrossoversbetweenhomologues,

mostoftheUV‐inducedrecombinationeventsarelikelytorepresentsister‐chromatid

interactions(KADYKandHARTWELL1992).

Relationshipbetweenmitoticgeneconversionandcrossovers:Inmeiosisinyeast,

abouthalfofconversioneventsareassociatedwithcrossovers(MANCERAetal.2008;PETES

etal.1991).Inourpreviousmitoticstudies,weselectedcrossoversandfoundthatmost

(>80%)oftheseeventswereassociatedwithanadjacenttractofgeneconversion(LEEetal.

2009;LEEandPETES2010);conversioneventsunassociatedwithcrossoverscouldnotbe

selectedwithoursystem.Inthecurrentstudy,forunselectedeventsstimulatedby

radiation,wecanestimatethefractionofconversioneventsthatareassociatedwith

crossovers.

ForIR‐treatedcells,includingallnon‐selectedeventsexceptBIR,wefoundfour

conversionsassociatedwithcrossoversandelevenconversionsunassociatedwith

crossovers(Tables2and3).Intheseexperiments,wedetecttheassociatedcrossover

becauseitgeneratesLOHfromtheconversiontracttotheendofthechromosome.As

showninFig.2,dependingonthepatternofchromosomesegregation,weexpectthatonly

halfofthecrossoverswillleadtoLOHofmarkersdistaltothepointofexchange,andthis

expectationhasexperimentalsupport(CHUAandJINKS‐ROBERTSON1991).Inaddition,as

discussedintheSupportingInformationandTableS12,wefoundpreliminaryevidencein

31

ourexperimentsforconversioneventsassociatedwithcrossoversthatdidnotresultin

LOH.

Thus,wecalculatethatofthefifteenconversioneventsinducedbyrays,itislikely

thateightwereassociatedwithcrossovers(53%association).Similarly,amongUV‐induced

recombinants,sincethereweresevenunselectedconversionsassociatedwithcrossovers

and33unselectedconversionsnotassociatedwithLOH(Tables2and3),wecalculatethat

about35%oftheUV‐inducedconversioneventsareassociatedwithcrossovers.Our

conclusionthatthefrequencyofcrossoversassociatedwithconversionsisnotvery

differentformitoticandmeioticconversioneventsisconsistentwithotherrecentstudies

(HOetal.2010).Inyeaststudiesinwhichconversioneventsarelimitedinsize,the

associationbetweenconversionandcrossoversisweaker(PÂQUESandHABER1999).Also,

inDrosophilaandmammaliancells,conversioneventsareonlyrarelyassociatedwith

crossovers(ANDERSENandSEKELSKY2010).

ComplexgeneconversiontractsandBIRevents:Previously,weclassified

conversiontractsas“simple”ifthemarkerswithinthetracthadoneofthefollowing

patterns:1)allmarkerswere3:1or1:3(notmixturesof3:1and1:3insametract),2)all

markerswere4:0or0:4,or3)hybridtractsoftheform3:1/4:0,1:3/0:4,3:1/4:0/3:1,or

1:3/0:4/1:3.Allsuchtractscanbeexplainedastheconsequenceoftherepairofoneortwo

brokenchromatidsbyoneoftheHRpathwaysshowninFig.1.Therewere,however,

conversiontractsthatweremorecomplicated(termed“complextracts”),whichwillbe

describedbelow.IntheUV‐treatedsamples,sixoftenofthecrossover‐associated

conversiontractswerecomplex,althoughonlythreeofthirty‐threetractswerecomplexin

conversionsunassociatedwithcrossovers(Tables2and3);thisdifferenceissignificant

32

(p<0.01)bytheFisherexacttest.IntheIR‐treatedsamples,theconversionevents

associatedwithcrossoverswereusuallymorecomplexthanthosethatwerenot(Table2),

althoughthedifferencewasnotsignificant.MANCERAetal.(MANCERAetal.2008)reported

that11%ofmeioticcrossovershadcomplexconversiontracts,whereasthefrequencyof

complextractsamongconversionsunassociatedwithcrossoverswas3%.Oneexplanation

ofthisdifferencecouldbethatcrossoversthatproceedthroughthepathwayshowninFig.

1Bareassociatedwithtworegionsofheteroduplex,whileconversionsresultingfromSDSA

ordHJdissolutionhaveonlyasingleregionofheteroduplex(Fig.1Aand1C).Second,

becausegeneconversiontractsassociatedwithcrossoversareusuallylongerthanthose

unassociatedwithcrossovers,theremaybeagreaterchancetoobservepatchyrepairof

mismatches(asdefinedbelow)intractsassociatedwithcrossovers.

DiagramsofallrecombinationeventsinourstudyareshowninTablesS1andS2,and

figuresshowingthepatternsofDSBrepairrequiredtoproducetherecombinationevents

areshowninFigs.S1‐S40.Mostofthecomplexconversiontractscouldbedividedintotwo

categories:thosethathadmultipletransitionsbetween3:1,4:0,andheterozygositywithin

thetractandthosetractsinwhich3:1and1:3or4:0and0:4segmentsoccurredwithinone

tract.Examplesofconversiontractswithmultipletransitionsarestrains18A(ClassJ9,

TableS1)and4.1(ClassJ8,TableS1);both18Aand4.1arealsodepictedinFig.5A.The

complextractin4.1isconsistentwiththerepairoftwoDSBswith“patchy”repairof

mismatchesintwooftheresultingheteroduplexes(Fig.S29).Heteroduplexeswilloften

containmultiplemismatchesthatcanberepairedtoproduceeitheraconversioneventora

restorationevent(KIRKPATRICKetal.1998).Forexample,inFig.1A,repairofthe

heteroduplexresultinginaduplexwithtwo“red”strandswouldrepresentconversion‐

33

typerepair,sincethispatternproduces3:1segregation;repairofthemismatchtoproduce

aduplexwithtwo“blue”strandsrepresentsrestoration‐typerepair,sincethispattern

generatestwocellsthatretainheterozygosityatthepositionoftheoriginalheteroduplex.

Althoughmultiplemismatcheswithinoneheteroduplexaregenerallyconvertedina

concertedmanneryieldingacontinuousconversiontract,tractswithmixturesof

conversion‐typeandrestoration‐typerepairhavebeendetectedinbothmeiosis(MANCERA

etal.2008;SYMINGTONandPETES1988)andmitosis(MITCHELetal.2010;NICKOLOFFetal.

1999).

ThepathwayofDSBrepairshowntoexplainthepatternofmarkersinthestrain18A

conversionevent(Fig.S30)invokespatchyrepairandbranchmigrationofthedHJ.During

recombinationinE.coli,aHollidayjunctioncanbetranslocatedbybranchmigration,

resultinginsymmetricheteroduplexes(WEST1997).Althoughgeneticevidenceargues

againsttheformationofsymmetricheteroduplexesduringmeioticrecombinationinS.

cerevisiae(PETESetal.1991),symmetricheteroduplexeshavebeeninvokedpreviouslyto

explaincertainclassesofmitoticgeneconversions(ESPOSITO1978;NICKOLOFFetal.1999;

ROITGRUNDetal.1993).Branchmigrationcanalsogeneratepatternsofrepairinwhicha

singleDSBcanproduceboth3:1and1:3,or4:0and0:4eventsasshowninFig.S10B.

Ourdatadonotallowustodetermineunambiguouslythepathwaysrequiredto

generatetheobservedconversiontracts.However,wecanstatethatmanyofthecomplex

tractsareinconsistentwiththesimplestformoftherecombinationmodelsshowninFig.1.

Inparticular,itislikelythatpatchyrepairofmismatchesisarelativelycommonfeatureof

mitoticgeneconversiontracts.Adetaileddiscussionofalloftheconversiontractsinour

studiesisgivenintheSupportingInformation.

34

TherewerethreeunselectedBIReventsobservedinourstudy(ClassL,Fig.S39and

S40).Fortwoofthethreeevents,weobservedaregionofconversionassociatedwiththe

BIRevent.ThispatternisconsistentwiththerepairoftwoDSBs,onebySDSAandoneby

BIR(Fig.S40).TheBIReventswereaboutthree‐foldlessfrequentthanunselected

crossovers,asexpectedfrompreviousstudies(HOetal.2010;MCMURRAYandGOTTSCHLING

2003)

RelationshipbetweenthelevelofDNAdamageandthefrequencyofLOHevents:

The100GydoseofIRusedinourexperimentsisexpectedtoproduceabout35

DSBs/diploidgenome(LEEandPETES2010).Sinceweobservedonly2.4LOH

events/irradiatedcell,mostoftheseDSBsmustberepairedbyamechanismthatdoesnot

produceadetectableLOHevent.SincethecellsinourexperimentswereirradiatedinG1,

theDSBsmusthavebeenrepairedeitherbyaninteractionwiththehomologous

chromosomeorbyNHEJ.Wesuggestseveralpossibleexplanations.First,itispossiblethat

therepairoftheDSBfrequentlyinvolvesaninteractionwiththehomologuethatis

associatedwithaveryshortconversiontract.Tractsshorterthan50bpwouldberarely

detected,evenbyHTS.Sucharepaireventwouldlikelyinvolveverylimitedprocessingof

brokenDNAendsaswellasshortexcisionrepairtracts.Asystemofshort‐patch(oftenless

than12basepairs)mismatchrepairthatisindependentoftheclassicalmismatchrepair

systeminS.cerevisiaewasdescribedbyCoicetal.(COICetal.2000),althoughthegenes

involvedinthistypeofrepairhavenotbeenidentified.Inaddition,conversiontractsless

than53bphavebeendetectedamongHO‐inducedevents(PALMERetal.2003).Arelated

possibilityisthatgeneconversioneventsoccurnon‐randomlyinregionsofthegenome

thatarenotrepresentedonourmicroarrays(regionsthatareidenticalbetweenW303a

35

andYJM789orregionswithrepeatedgenes).Athirdpossibilityisthattherepairofthe

DSBisassociatedwithrestoration‐typerepairofmismatcheswithintheheteroduplexes.

Sincemostofthecrossoversinourstudyareassociatedwithdetectablegeneconversion

tracts,wewouldhavetohypothesizethatconversioneventsthatarenotassociatedwith

crossoversaremuchmorepronetorestoration‐typerepairthanconversioneventsthatare

associatedwithcrossovers.AfourthpossibilityisthattheIR‐inducedDSBsarefrequently

(DALEYetal.2005)repairedbyNHEJevents.AlthoughNHEJeventsarerepressedin

MATa/MATdiploids,sincePG311lackstheMATlocus,NHEJeventswilloccur.Although

NHEJeventswillnotproduceLOH,dependingonthenatureofDNAends(compatible

single‐strandoverhangsorblunt),someNHEJeventswouldbeexpectedtoresultinlossor

gainofafewbasepairs.Althoughwedidnotobservein/delsinourHTSanalysis,this

observationdoesnotruleoutthepossibilitythatsomerepaireventsreflectNHEJ.Itisalso

possible,ofcourse,thatallfourpossibilitiesdescribedabovearepartlyresponsibleforthe

“missing”LOHevents.

Oneexplanationthatwecanexcludeasamajorcontributortothediscrepancy

betweenthenumberoflesionsandthenumberofLOHeventsischromosomeloss.

ChromosomelosscanbereadilydetectedbytheSNPmicroarrays,andnolosseswere

observedincellstreatedwithγraysorUV.Inexperimentsinwhicheight‐foldhigherdoses

ofγrayswereused,about10%ofthetreatedyeastcellshadchromosomeloss(ARGUESOet

al.2008).

Althoughwedetectedmorethan50unselectedLOHeventsincellstreatedwithγrays

andUV,noduplicationsordeletionsweredetected.Thus,SNParraysthatcandetectboth

LOHandchangesincopynumberareamuchmoreefficientmethodofdetecting

36

recombinogenicDNAlesionsthancomparativegenomichybridization(CGH)arrays.Inour

previousanalysisofγray‐treateddiploidcellsbyCGH(ARGUESOetal.2008),wefoundthat

mostoftheirradiatedcellshadoneormorechromosomerearrangements,usuallynon‐

reciprocaltranslocationswithretrotransposonsatthebreakpoints.Intheseexperiments,

wetreatedG2‐synchronizedcellswithdosesofradiationthatwereeight‐foldhigherthan

thedosesusedinourcurrentstudy.

MutationsinducedbyγraysandUV:Wefoundonlyafewmutationsinducedbyγ

raysandUV,averagesof1.5and6mutations/irradiatedcell,respectively.Althoughthere

arenogenome‐widestudiesofthefrequenciesofmutationsinducedbyγrays,

extrapolatingfromthefrequencyofinductionofX‐ray‐inducedmutationsattheCAN1

locus(GOEKEandMANNEY1979)andtherateofspontaneousmutationsperbasepairat

CAN1(LANGandMURRAY2008),wecalculatethattheexpectedfrequencyofmutationsper

genomeisabouttwo/diploidcell,closetoourobservednumber.Themostdirect

comparisonfortheUV‐inducedmutationsiswithdataobtainedfromHTSofUV‐treated

stationary‐phasehaploidyeaststrains(BURCHetal.2011).Thesestrainsinthesestudies

hadatemperature‐sensitivemutationinCDC13.However,threeofthesequencedisolates

weretreatedatthepermissivetemperature.ByextrapolatingtheirdatatoourUVdose,we

wouldexpectabout14mutations/diploidcell,onlytwo‐folddifferentfromourobserved

frequencies.Insummary,ourHTSdatadetectedroughlytheexpectednumberofmutations

perirradiatedstrain.

AsdescribedintheResults,themutationsinducedbyUVandγraysarenon‐randomly

distributedamongtheyeastchromosomes.Althoughthisnon‐randomdistributionneeds

tobeverifiedwithalargedataset,itispossiblethatthemutagenicDNAdamageis

37

distributednon‐randomlybecauseofthespecificpositionofdifferentchromosomeswithin

thenucleusorchromosome‐specificchromatindomains.SincetheUV‐irradiatedstrains

haveabout7500DNAlesions(asdiscussedabove),thevastmajorityoftheselesionsmust

berepairedbynucleotideexcisionrepairinamannerthatdoesnotresultinLOHor

mutations.

WeassumethatmostoftheUV‐inducedmutationsreflecterrorsintroducedduringthe

bypassofpyrimidinedimersbyRev1pandPol, since90%ofUV‐inducedmutations

requiretheseactivities(LAWRENCE2002).Thesourceofthemutationsintheγ‐ray‐treated

samplesislessclear.SincethemutationsarenotassociatedwithregionsofLOH,the

mutationsprobablydonotreflecterrorsintroducedbyDSBrepair.Itispossiblethatbases

damagedbyγraysarebypassedbyerror‐pronepolymerasesbyamechanismsimilarto

thatassociatedwithUV‐inducedDNAdamage.

Allthreeofthemutationsintroducedbyγirradiationandabouthalfofthemutations

causedbyUVwerefoundinbothsectorsofsectoredcolonies.Thisresultindicatesthatthe

introducedmutationwasplacedintobothstrandsoftheduplexbeforeDNAreplication.

Suchevents,whichhavebeenobservedpreviouslyforUV‐inducedDNAdamage(ECKARDT

andHAYNES1977;JAMESandKILBEY1977),havebeentermed“two‐strand”mutations

(ABDULOVICetal.2006).Onemodelforsucheventsisthattheyreflecttherepairoftwo

closely‐opposedDNAlesionsbynucleotideexcisionrepair(NER).Duringtherepairofone

lesion,amutationisintroduced.Therepairofthesecondlesionontheoppositestrand

resultsinagapthatincludesthemutantsubstitution,andfilling‐inofthegapresultsin

mutantsubstitutionsinbothstrandsoftheduplex.Whatevertheexplanationoftwo‐strand

events,bothUVandγraysefficientlyproducethistypeofmutation.

38

TherepairofDSBsisassociatedwitha100‐foldelevationinthefrequencyofreversion

ofaclosely‐linkedmutation(STRATHERNetal.1995),andapproximately1000‐foldelevated

ratesofmutationhavebeenobservedduringBIR(DEEMetal.2011)andothergene

conversioneventsthatresultintwonewly‐synthesizedstrands(HICKSetal.2010).In

addition,thefrequencyofUV‐inducedmutagenesisiselevatedmorethan100‐foldin

regionsofsingle‐strandedDNAnexttoDSBsorabnormaltelomeres(YANGetal.2008).

Basedontheseobservations,wecheckedwhetherthedenovomutationswerenon‐

randomlyassociatedwithLOHregionsassociatedwithgeneconversionorBIR.Therewere

15basesubstitutionsobservedamongfoursectoredcoloniesresultingfromtheirradiation

ofG1cells.ThetotallengthsoftheunselectedgeneconversionandBIReventsamongthese

strainswere163kb(PG311‐UV‐8R/W),271kb(PG311‐UV‐9R/W),22kb(PG311‐IR‐

37R/W),and50kb(PG311‐IR‐40R/W).ThefractionofthegenomewiththeseLOHregions

wasabout0.01.Twoofthefifteen(0.13)mutationswereinregionsofLOH.Althoughthis

calculationsuggeststhattheLOHregionsmayhaveasignificantlyelevatedfrequencyof

mutations,mostoftheinducedmutationsarelocatedoutsideoftheLOHregions.

Comparisonamongmethodsofphysicallymappingrecombinationevents:Inour

previousstudies,wemappedrecombinationeventsbyaPCR‐basedtechnique(described

intheIntroduction).AsemployedinouranalysisofchromosomeVevents,thisapproach

wastime‐consumingandexpensive,andmappedeventswithrelativelypoorresolution

(about4kb).Moreimportantly,thismethodcouldnotbeeasilyusedtomapevents

throughoutthegenome.Inaddition,thePCR‐basedapproachdidnotallowustoexamine

changesingenedosage(deletionsorduplications).Forexample,wefoundthatanevent

39

classifiedasacrossoveronchromosomeVbythePCR‐basedmethodwasactuallya

terminaldeletiononVwhenexaminedbySNParrays.

Incontrast,bothSNParraysandHTSallowanalysisofeventsthroughoutthegenome.

TheadvantagesofSNParrayscomparedtoHTSare:1)relativelylowcost(about

$100/sample),2)speedofanalysis(aboutfourhoursforSNParraysversusaweekfor

HTS),and3)relativeeaseindetectingchangesingenedosage.Themajoradvantagesof

HTSare:1)higherresolution(1kbforSNParraysversus250bpforHTS),and2)the

abilitytodetectdenovomutations.Inaddition,diagnosisofLOHbyHTScanbedonewith

anydiploidinwhichtheprogenitorhaploidstrainshavebeensequenced,whereas

diagnosisofLOHbySNParraysrequirestheconstructionofstrain‐specificmicroarrays.

AlthoughSNParraysareprobablyamorecost‐effectiveandfasterapproachformapping

largenumbersofrecombinationeventsatpresent,asHTSbecomescheaperandanalysisof

HTSdatabecomesfaster,HTSislikelytobethemethodofchoiceinthefuture.NeitherSNP

microarraysnorHTS,however,canmaprecombinationeventsthatdonotinvolveLOH(for

example,sisterchromatidexchanges).

Summary:Inconclusion,wehaveusedSNPmicroarraysandHTStomapcrossovers

andgeneconversioneventsathighresolutionthroughouttheyeastgenome.Thesestudies

representthefirstgenome‐widemeasurementofthenumberandtypesofunselectedLOH

eventsinducedbyUVandrays.InG1‐synchronizedcellstreatedwitheitherUVorrays,

4:0conversioneventsarecommon,suggestingthatmanyoftheLOHeventsreflectthe

repairoftwosisterchromatidsbrokenatapproximatelythesameposition.Inaddition,the

high‐resolutionanalysisofrecombinationeventsbySNParraysandHTSrevealsthatgene

40

conversiontracts,particularlythoseassociatedwithcrossovers,aremorecomplexthan

waspreviouslyrecognizedbylow‐resolutionstudies.

41

ACKNOWLEDGMENTS

WewouldliketothankmembersofthePETESandJINKS‐ROBERTSONlabsfor

discussions,andLUCASARGUESO,KATMITCHEL,SUEJINKS‐ROBERTSON,andWEISONGfor

commentsonthemanuscript.WethankmembersofJ.MCCUSKER’Slabfortechnicalhelp,

andL.ARGUESOandJ.RINEforusefulsuggestions. TheresearchwassupportedbyNIHgrants

GM24110(TDP),GM52319(TDPandPM),5RC1ES18091(TDPandPM),andfundsfrom

theDukeUniversitySchoolofMedicine.

42

REFERENCES

ABBEY,D.,M.HICKMAN,D.GRESHAMandJ.BERMAN,2011High‐resolutionSNP/CGHmicroarrays

revealtheaccumulationoflossofheterozygosityincommonlyusedCandidaalbicans

strains.Genes/Genomes/Genetics1:523‐530.

ABDULOVIC,A.,N.KIMandS.JINKS‐ROBERTSON,2006MutagenesisandthethreeR'sinyeast.DNA

Repair(Amst)5:409‐421.

AGUILERA,A.,2002Theconnectionbetweentranscriptionandgenomicinstability.EMBOJ21:195‐

201.

AGUILERA,A.,andH.L.KLEIN,1989Yeastintrachromosomalrecombination:longgeneconversion

tractsarepreferentiallyassociatedwithreciprocalexchangeandrequiretheRAD1and

RAD3geneproducts.Genetics123:683‐694.

ALLERS,T.,andM.LICHTEN,2001Differentialtimingandcontrolofnoncrossoverandcrossover

recombinationduringmeiosis.Cell106:47‐57.

ALTMAN,D.G.,1990PracticalStatisticsforMedicalResearch.CRC,NewYork.

ANDERSEN,S.L.,andJ.SEKELSKY,2010Meioticversusmitoticrecombination:twodifferentroutesfor

double‐strandbreakrepair:thedifferentfunctionsofmeioticversusmitoticDSBrepairare

reflectedindifferentpathwayusageanddifferentoutcomes..Bioessays32:1058‐1066.

ARGUESO,J.L.,J.WESTMORELAND,P.A.MIECZKOWSKI,M.GAWEL,T.D.PETESetal.,2008Double‐strand

breaksassociatedwithrepetitiveDNAcanreshapethegenome.ProcNatlAcadSciUSA

105:11845‐11850.

BARBERA,M.A.,andT.D.PETES,2006Selectionandanalysisofspontaneousreciprocalmitoticcross‐

oversinSaccharomycescerevisiae.ProcNatlAcadSciUSA103:12819‐12824.

BRANZEI,D.,andM.FOIANI,2010Maintaininggenomestabilityatthereplicationfork.NatRevMol

CellBiol11:208‐219.

43

BREEN,A.P.,andJ.A.MURPHY,1995ReactionsofoxylradicalswithDNA.Freeradicalbiology&

medicine18:1033‐1077.

BURCH,L.H.,Y.YANG,J.F.STERLING,S.A.ROBERTS,F.G.CHAOetal.,2011Damage‐inducedlocalized

hypermutability.CellCycle10:1‐13.

CHUA,P.,andS.JINKS‐ROBERTSON,1991Segregationofrecombinantchromatidsfollowingmitotic

crossingoverinyeast.Genetics129:359‐369.

COIC,E.,L.GLUCKandF.FABRE,2000Evidenceforshort‐patchmismatchrepairinSaccharomyces

cerevisae.EmboJ19:3408‐3417.

DAIGAKU,Y.,A.A.DAVIESandH.D.ULRICH,2010Ubiquitin‐dependentDNAdamagebypassis

separablefromgenomereplication.Nature465:951‐955.

DALEY,J.M.,P.L.PALMBOS,D.WUandT.E.WILSON,2005Nonhomologousendjoininginyeast.Annu

RevGenet39:431‐451.

DEEM,A.,A.KESZTHELYI,T.BLACKGROVE,A.VAYL,B.COFFEYetal.,2011Break‐inducedreplicationis

highlyinaccurate.PLoSBiology9:e1000594.

ECKARDT,F.,andR.H.HAYNES,1977Inductionofpureandsectoredmutantclonesinexcision‐

proficientanddeficientstrainsofyeast.MutatRes43:327‐338.

ESPOSITO,M.S.,1978Evidencethatspontaneousmitoticrecombinationoccursatthetwo‐strand

stage.ProcNatlAcadSciUSA75:4436‐4440.

FABRE,F.,1978InducedintragenicrecombinationinyeastcanoccurduringtheG1mitoticphase.

Nature272:795‐798.

FACHINETTI,D.,R.BERMEJO,A.COCITO,S.MINARDI,Y.KATOUetal.,2010Replicationterminationat

eukaryoticchromosomesismediatedbyTop2andoccursatgenomiclocicontaining

pausingelements.MolCell39:595‐605.

44

FRANKLIN,W.A.,P.W.DOETSCHandW.A.HASELTINE,1985Structuraldeterminationoftheultraviolet

light‐inducedthymine‐cytosinepyrimidine‐pyrimidone(6‐4)photoproduct.Nucleicacids

research13:5317‐5325.

FRIEDBERG,E.C.,G.C.WALKER,W.SIEDE,R.D.WOOD,R.A.SCHULTZetal.,2006DNARepairand

Mutagenesis.ASMPress,WashingtonDC.

GALLI,A.,andR.H.SCHIESTL,1999Celldivisiontransformsmutageniclesionsintodeletion‐

recombinogeniclesionsinyeastcells.MutatRes429:13‐26.

GOEKE,E.,andT.R.MANNEY,1979Expressionofradiation‐inducedmutationsatthearginine

permease(CAN1)locusinSaccharomycescerevisiae.Genetics91:53‐66.

GRESHAM,D.,B.CURRY,A.WARD,D.B.GORDON,L.BRIZUELAetal.,2010Optimizeddetectionof

sequencevariationinheterozygousgenomesusingDNAmicroarrayswithisothermal‐

meltingprobes.ProcNatlAcadSciUSA107:1482‐1487.

GUTHRIE,C.,andG.R.FINK,1991GuidetoYeastGeneticsandMolecularBiology.AcademicPress,San

Diego.

HEYER,W.D.,K.T.EHMSENandJ.LIU,2010Regulationofhomologousrecombinationineukaryotes.

AnnuRevGenet44:113‐139.

HICKS,W.M.,M.KIMandJ.E.HABER,2010Increasedmutagenesisanduniquemutationsignature

associatedwithmitoticgeneconversion.Science329:82‐85.

HO,C.K.,G.MAZON,A.F.LAMandL.S.SYMINGTON,2010Mus81andYen1promotereciprocal

exchangeduringmitoticrecombinationtomaintaingeneticintegrityinbuddingyeast.Mol

Cell40:10077‐10082.

JAMES,A.P.,andB.J.KILBEY,1977ThetimingofUVmutagenesisinyeast:apedigreeanalysisof

inducedrecessivemutation.Genetics87:237‐248.

JESSOP,L.,T.ALLERSandM.LICHTEN,2005Infrequentco‐conversionofmarkersflankingameiotic

recombinationinitiationsiteinSaccharomycescerevisiae.Genetics169:1353‐1367.

45

KADYK,L.C.,andL.H.HARTWELL,1992Sisterchromatidsarepreferredoverhomologsassubstrates

forrecombinationalrepairinSaccharomycescerevisiae.Genetics132:387‐402.

KIRKPATRICK,D.T.,M.DOMINSKAandT.D.PETES,1998Conversion‐typeandrestoration‐typerepair

ofDNAmismatchesformedduringmeioticrecombinationinSaccharomycescerevisiae.

Genetics149:1693‐1705.

KUNZ,B.A.,M.K.PIERCE,J.R.MISandC.N.GIROUX,1987DNAsequenceanalysisofthemutational

specificityofu.v.lightintheSUP4‐ogeneofyeast.Mutagenesis2:445‐453.

LANG,G.I.,andA.W.MURRAY,2008Estimatingtheper‐base‐pairmutationrateintheyeast

Saccharomycescerevisiae.Genetics178:67‐82.

LAWRENCE,C.W.,2002CellularrolesofDNApolymerasezetaandRev1protein.DNARepair(Amst)

1:425‐435.

LEE,P.S.,P.W.GREENWELL,M.DOMINSKA,M.GAWEL,M.HAMILTONetal.,2009Afine‐structuremapof

spontaneousmitoticcrossoversintheyeastSaccharomycescerevisiae.PLoSGenet5:

e1000410.

LEE,P.S.,andT.D.PETES,2010FromtheCover:mitoticgeneconversioneventsinducedinG1‐

synchronizedyeastcellsbygammaraysaresimilartospontaneousconversionevents.Proc

NatlAcadSciUSA107:7383‐7388.

LEMOINE,F.J.,N.P.DEGTYAREVA,K.LOBACHEVandT.D.PETES,2005Chromosomaltranslocationsin

yeastinducedbylowlevelsofDNApolymeraseamodelforchromosomefragilesites.Cell

120:587‐598.

LINDBLAD‐TOH,K.,D.M.TANENBAUM,M.J.DALY,E.WINCHESTER,W.O.LUIetal.,2000Loss‐of‐

heterozygosityanalysisofsmall‐celllungcarcinomasusingsingle‐nucleotidepolymorphism

arrays.NatBiotechnol18:1001‐1005.

MANCERA,E.,R.BOURGON,A.BROZZI,W.HUBERandL.M.STEINMETZ,2008High‐resolutionmappingof

meioticcrossoversandnon‐crossoversinyeast.Nature454:479‐485.

46

MCCULLEY,J.L.,andT.D.PETES,2010Chromosomerearrangementsandaneuploidyinyeaststrains

lackingbothTel1pandMec1preflectdeficienciesintwodifferentmechanisms.ProcNatl

AcadSciUSA107:11465‐11470.

MCMURRAY,M.A.,andD.E.GOTTSCHLING,2003Anage‐inducedswitchtoahyper‐recombinational

state.Science301:1908‐1911.

MERKER,J.D.,M.DOMINSKAandT.D.PETES,2003Patternsofheteroduplexformationassociatedwith

theinitiationofmeioticrecombinationintheyeastSaccharomycescerevisiae.Genetics165:

47‐63.

MITCHEL,K.,H.ZHANG,C.WELZ‐VOEGELEandS.JINKS‐ROBERTSON,2010Molecularstructuresof

crossoverandnoncrossoverintermediatesduringgaprepairinyeast:implicationsfor

recombination.MolCell38:211‐222.

NAKAI,S.,andR.K.MORTIMER,1969Studiesofthemechanismofradiation‐inducedmitotic

segregationinyeast.MolGenGenet103:329‐338.

NICKOLOFF,J.A.,D.B.SWEETSER,J.A.CLIKEMAN,G.J.KHALSAandS.L.WHEELER,1999Multiple

heterologiesincreasemitoticdouble‐strandbreak‐inducedallelicgeneconversiontract

lengthsinyeast.Genetics153:665‐679.

PALMER,S.,E.SCHILDKRAUT,R.LAZARIN,J.NGUYENandJ.A.NICKOLOFF,2003Geneconversiontractsin

Saccharomycescerevisiaecanbeextremelyshortandhighlydirectional.NucleicAcidsRes

31:1164‐1173.

PÂQUES,F.,andJ.E.HABER,1999Multiplepathwaysofrecombinationinducedbydouble‐strand

breaksinSaccharomycescerevisiae.MicrobiolMolBiolRev53:394‐404.

PEAK,M.J.,andJ.G.PEAK,1986DNA‐to‐proteincrosslinksandbackbonebreakscausedbyfar‐and

near‐ultraviolet,andvisiblelightradiationsinmammaliancells.Basiclifesciences38:193‐

202.

47

PETES,T.D.,andC.W.HILL,1988Recombinationbetweenrepeatedgenesinmicroorganisms.Annu

RevGenet22:147‐168.

PETES,T.D.,R.E.MALONEandL.S.SYMINGTON,1991Recombinationinyeast,pp.407‐521inThe

MolecularandCellularBiologyoftheYeastSaccharomyces,editedbyJ.R.BROACH,JONES,E.W.,

ANDJ.R.PRINGLE.ColdSpringHarborPress,ColdSpringHarbor.

REYNOLDS,R.J.,1987InductionandrepairofcloselyopposedpyrimidinedimersinSaccharomyces

cerevisiae.MutatRes184:197‐207.

ROITGRUND,C.,R.STEINLAUFandM.KUPIEC,1993Donationofinformationtotheunbroken

chromosomeindouble‐strandbreakrepair.CurrGenet23:414‐422.

SETLOW,R.B.,1966Cyclobutane‐typepyrimidinedimersinpolynucleotides.Science153:379‐386.

SHKUNDINA,I.S.,andM.D.TER‐AVANESYAN,2007Prions.Biochemistry(Moscow)72:19‐36.

SHRIVASTAV,M.,L.P.DEHAROandJ.A.NICKOLOFF,2008RegulationofDNAdouble‐strandbreak

repairpathwaychoice.CellRes18:134‐147.

STRATHERN,J.N.,B.K.SHAFERandC.B.MCGILL,1995DNAsynthesiserrorsassociatedwithdouble‐

strand‐breakrepair.Genetics140:665‐672.

SYMINGTON,L.S.,andT.D.PETES,1988Expansionsandcontractionsofthegeneticmaprelativeto

thephysicalmapofyeastchromosomeIII.MolCellBiol8:595‐604.

TANG,W.,M.DOMINSKA,P.W.GREENWELL,J.HARVENEK,K.LOBACHEVetal.,2011Friedreich'sAtaxia

(GAA)/(TTC)repeatsstronglystimulatemitoticcrossoversinSaccharomycescerevisiae.

PLoSGenet7:e1001270.

WARD,J.F.,1990TheyieldofDNAdouble‐strandbreaksproducedintracellularlybyionizing

radiation:areview.Internationaljournalofradiationbiology57:1141‐1150.

WEI,W.,J.H.MCCUSKER,R.W.HYMAN,T.JONES,Y.NINGetal.,2007Genomesequencingand

comparativeanalysisofSaccharomycescerevisiaestrainYJM789.ProcNatlAcadSciUSA

104:12825‐12830.

48

WEST,S.C.,1997ProcessingofrecombinationintermediatesbytheRuvABCproteins.AnnuRev

Genet31:213‐244.

WINZELER,E.A.,C.I.CASTILLO‐DAVIS,G.OSHIRO,D.LIANG,D.R.RICHARDetal.,2003Geneticdiversityin

yeastassessedwithwhole‐genomeoligonucleotidearrays.Genetics163:79‐89.

YANG,Y.,J.STERLING,F.STORICI,M.A.RESNICKandD.A.GORDENIN,2008Hypermutabilityofdamaged

single‐strandDNAformedbydouble‐strandbreaksanduncappedtelomeresinyeast

Saccharomycescerevisiae.PLoSGenet4:e1000264.

49

Figurelegends

Figure1.PathwaysofDSBrepairbyhomologousrecombination.

Inthisfigure,weshowacceptedmodelsofDSBrepairbyhomologousrecombination.

DNAstrandsfromtwodifferenthomologuesareshowninredandblue;lightredandblue

linesindicatenewly‐synthesizedDNA.Regionsoftheduplexthathavestrandsofdifferent

colorsrepresentheteroduplexes.Thesepathwaysaredescribedindetailinthetext.

A.Synthesis‐dependentstrandannealing(SDSA)pathway.Followingprocessingofthe

DSB,the3’endoftheleftendofthebrokenDNAmoleculeinvadestheotherduplex.

FollowingDNAsynthesis,theinvadingstrandisdisplaced,andhybridizestotherightend

ofthebrokenchromosome.Thispathwayresultsinconversioneventsunassociatedwith

crossovers.

B.Double‐strandbreakrepair(DSBR)pathway.Inthispathway,adoubleHolliday

junction(dHJ)isformed.Resolutionofthesejunctionsbyresolvasecleavagecanresultin

twodifferentcrossoverproducts(middlepanel)andtwodifferentnon‐crossoverproducts

(rightpanel).Theseproductshavetworegionsofheteroduplexlocatedintrans.

Alternatively,thedHJcanbedissolvedbytheactionoftopoisomerases/helicasesresulting

inanon‐crossoverproductwithheteroduplexeslocatedincis.

C.Break‐inducedreplication(BIR)pathway.Oneofthebrokenendsinvadesthe

homologouschromosome,andduplicatessequencesfromthepointofinvasiontothe

telomere.ThenetresultofBIReventsisanapparentlongterminalgeneconversionevent.

Figure2.Geneticsystemusedtoselectmitoticcrossoversandassociatedconversions

ontheleftarmofchromosomeV.

50

ThestartingdiploidstrainPG311hastheochre‐suppressiblecan1‐100ononecopyof

chromosomeV(showninred)andtheSUP4‐ogene(encodinganochresuppressortRNA

gene)atanallelicpositionontheotherhomologue(showninblack).Thestrainis

homozygousfortheochre‐suppressibleade2‐1allele.Strainswithanunsuppressedade2‐1

mutationformredcolonies.Thestartingdiploidstrainiscanavanine‐sensitiveandforms

pinkcolonies.

A.ReciprocalcrossoverwithoutanassociatedgeneconversioninitiatedbyasingleDSB

inG2.Thistypeofeventproducesacanavanine‐resistantred/whitesectoredcolony

(BARBERAandPETES2006).ThetransitionfromheterozygousmarkerstoLOHisidenticalin

thetwosectors.

B.ReciprocalcrossoverwithanassociatedconversioneventinitiatedbyasingleDSBin

G2.IfaDSBformsononeoftheblackchromatids,aconversionassociatedwiththe

crossovermayoccur.Thiseventwillalsoresultinacanavanine‐resistantred/white

sectoredcolonyinwhichthetransitionsbetweenheterozygousmarkersandLOHare

differentinthetwosectors.Theregionofconversionisindicatedbythebluerectangle.

C.ReciprocalcrossoverandconversionresultingfromaDSBformedinG1.Ablack

chromosomewithaDSBisreplicatedtoformtwosisterchromatidsthatarebrokenatthe

sameplace.Onechromatidisrepairedtoyieldareciprocalcrossoverandanassociated

conversion;thesecondisrepairedtoyieldaconversionwithoutacrossover.Theresulting

redandblacksectorswillhavea4:0conversionevent,aregioninwhichbothsectorsare

homozygousforSNPsderivedfromtheredchromatid(includedwithinthebluerectangle).

51

Figure3.Productionofhybridconversiontractsbyrepairoftwobrokensister

chromatids.TheblackchromosomeisbrokeninG1andreplicatedtoyieldtwobroken

sisterchromatids.

A.Productionofa3:1/4:0hybridtract.IfthetwoDSBsarerepairedtoyield

conversiontractsthathavethesamecentromere‐proximalboundary,butdifferent

centromere‐distalboundaries,a3:1/4:0hybridwillbegenerated(shownintheblue

rectangle).

B.Productionofa3:1/4:0/3:1hybridtract.Ifoneconversioneventisextendedbeyond

theotheratboththecentromere‐proximalandcentromere‐distalboundaries,a

3:1/4:0/3:1tractwillbeformed.

Figure4.Analysisofaspontaneousreciprocalcrossover(PG311‐2A)ontheleftarmof

chromosomeVbySNPmicroarrays.Mostofthedetailsconcerningthisfigureareexplained

inthetext.Inbrief,DNAsamplesisolatedfromtheredandwhitesectorswerelabeledwith

onefluorescentnucleotideandDNAfromaheterozygouscontrolstrainwaslabeledwitha

differentfluorescentnucleotide.ThesampleswerecompetitivelyhybridizedtotheSNP

arrayandwemeasuredtheratioofhybridizationoftheprobestoSNP‐specific

oligonucleotides.TheredandbluecolorsindicatehybridizationtotheW303a‐and

YJM789‐specificoligonucleotides,respectively.CEN5islocatedapproximatelyatSGD

coordinate152000.

A.Low‐resolutiondepictionofthesamplesderivedfromtheredandwhitesectors.In

theboxedregion,theredsectorhasaregionofLOHwhereasthewhitesectoris

heterozygousatthesameposition.Thispatternindicatesa3:1conversionassociatedwith

52

thecrossover.Centromere‐distaltotheconversionevent,theredandwhitesectorsare

homozygousfortheW303a‐andYJM789‐specificSNPs,respectively.

B.High‐resolutiondepictionofthesamplesderivedfromtheredandwhitesectors.

Eachblueandredsquarerepresentshybridizationtoasingleoligonucleotideonthearray;

theconvertedregionisboxed.

Figure5.Mappingofcrossoversandassociatedconversioneventsontheleftarmof

chromosomeVinuntreatedcells,andcellstreatedwithraysorUVbySNPmicroarrays.

Eachred/whitesectoredcanavanine‐resistantcolonyisgivenanumberandis

depictedasapairoflineswiththeupperlinerepresentingtheredsectorandthelowerline

showingthewhitesector.Thecoloredsegmentssignifyheterozygosityforthemarkers

(green),homozygosityfortheYJM789‐derivedSNPs(black),orhomozygosityforthe

W303a‐derivedSNPs(red).Thegreenarrowsshowthatthemarkersareheterozygous

fromthepositionthatthegreensegmentbeginstotheendofthechromosome,andthered

andblackarrowsindicatethatthemarkersarehomozygousfortheW303a‐ortheYJM789‐

derivedSNPs,respectively,fromthepointthatthesegmentbeginstothetelomereofthe

chromosome.Internalregionsofheterozygosityandhomozygosityareshownasline

segmentswithoutarrowsandaredrawnapproximatelytoscale.Thenumbersatthetopof

thefigureareSGDcoordinatesandtheregionbetweenCEN5andthecan1‐100/SUP4‐o

markersisabout120kbinlength.

A.Spontaneouscrossoversandconversions.

B.ray‐inducedcrossoversandconversions.

C.UV‐inducedcrossoversandconversions.

53

Figure6.Genomiclocationsofunselectedrecombinationeventsanddenovomutations

inuntreatedcellsandincellstreatedwithUVorraysasdeterminedbySNPmicroarrays

andHTS.

Thehorizontalblackbarsdepicteachchromosomeandareproportionaltothe

chromosomelengthexceptforchromosomeXII.Theblackcirclesdepictthecentromereof

eachchromosome.Shorthorizontalbarsaboveeachchromosomedepictconversionevents

unassociatedwithcrossoversandthelengthofeachbarisapproximatelyproportionalto

thelengthoftheconversiontract.Allconversiontractsareshownassinglesolidlines

withoutregardtothecomplexityoftheevent(forexample,transitionsbetween4:0and

3:1).SinglearrowheadsdepictreciprocalcrossoversanddoublearrowheadsdepictBIR

events.Asteriskslocatedonthechromosomeindicatetheapproximatepositionsof

mutationsinducedbyUVorrays;twoofthemutations(locatedatSGDcoordinates

171529and301552onX)areinregionsofLOH.Eventsobservedinuntreatedcells,cells

treatedwithUV,andcellstreatedwithraysareshowningreen,red,andblue,

respectively.NoneoftheeventsselectedontheleftarmofVareshowninthisfigure.

Figure7.SNParrayanalysisofageneconversioneventunassociatedwithacrossover.

Incellstreatedwithrays,oneofthecanavanine‐resistantred/whitesectored

colonies(43RW)hadanunselectedgeneconversioneventonchromosomeIV.Asshownat

low‐(Fig.7A)andhigh‐(Fig.7B)resolution,bothsectorshadanLOHregioninwhich

YJM789‐derivedSNPsbecamehomozygous(0:4conversionevent).Thedepictionofthe

SNParraydataisthesameasinFig.4.Thelengthoftheconversiontractisabout3kb.

CEN4islocatedapproximatelyatSGDcoordinate450000.

Figure8.AnalysisofthesamerecombinationeventbybothSNParraysandHTS.

54

ThisfigureshowstheanalysisoftheunselectedrecombinationeventonchromosomeII

intheredsectoroftheUV‐inducedsectoredcolony8.OurstandardSNParrayanalysis(top

partsofFig.8Aand8B)showedasingletransitionbetweenheterozygosityand

homozygosityataboutSGDcoordinate452000.Inthebottompanelsofthefigure,we

showHTSdataforthesamegenomicsample.FortheHTSdata,theYaxisrepresentsthe

frequencyofYJM789SNP(blue)orW303aSNP(red)“reads”fortheexperimentalsample

whenassembledtothePSL2genome.Forheterozygousregions,thereshouldbe

approximatelyequalfrequenciesofthetwotypesofSNPs.Itisclearfromthehigh‐

resolutiondepictionsoftheHTSdatathatthereisashortLOHregion(boxed)locatednear

SGDcoordinate450000thatwasnotdetectedbytheSNParrays.Thisregionwasnot

detectedbecauseoligonucleotidescontainingtheseSNPswerenotpresentonthearray.In

thelow‐resolutiondepictionoftheHTSdata,withintheLOHregion,thereisasmallregion

nearSGDcoordinate800000inwhichSNPsappeartobeheterozygous.Thesesignalsare

artifactsbasedon“reads”fromtherepeateddivergedMALandMPHgenesthatwere

incorrectlymappedbythegenomeanalysissoftwaretochromosomeII.CEN2islocated

nearSGDcoordinate238000.

TABLE 1. COMPARISON OF MAPPING METHODS FOR FOUR SPONTANEOUS CROSSOVERS ON CHROMOSOME V.

PCR-based method

SNP microarrays

Strain name Cen-prox.

coordinate

Cen-dist.

coordinate

Event description

Cen-prox.

coordinate

Cen-dist.

coordinate

Event description

PG311-1.4

133080

125754

CO, no conversion

130096

127038

CO + 3:1 tract

PG311-1.7

151440

146855

CO, no conversion

151419

150291

CO, no conversion

PG311-4.1

99267

60163

CO + hybrid tract

98763

62494

CO + complex tract

PG311-4.11

133080

94329

CO + hybrid tract

129511

97792

CO + hybrid tract

56

TABLE 2. SUMMARY OF ALL CROSSOVERS DIAGNOSED BY SNP MICROARRAYa

Types of reciprocal

crossovers Spontaneous -ray UV

Selected Unselected Selected Unselected Selected Unselected

No detectable conversions 2 0 0 0 0 0

3:1 or 1:3 conversions 2 0 0 1 0 2

4:0 or 0:4 conversions 1 0 1 0 0 0

Hybrid conversionsb 1 0 2 3 2 0

Complex conversions 7 0 4 0 1 5

Total crossovers 13 0 7 4 3 7

aIn this table, we summarize data from selected crossovers and associated conversion events on the left arm of

chromosome V as well as unselected crossovers and associated conversions on other chromosomes. For this

table, the data obtained with high-throughput DNA sequencing was not used.

b 3:1/4:0, 3:1/4:0/3:1, 1:3/0:4, or 1:3/0:4/1:3 conversion events.

57

TABLE 3. SUMMARY OF ALL UNSELECTED CONVERSION EVENTS UNASSOCIATED WITH LOH OR BIR

EVENTS AS DIAGNOSED BY SNP MICROARRAYS.

 

Type of event Spontaneous -ray UV

3:1 or 1:3 conversions 0 4 9

4:0 or 0:4 conversions 1 4 12

Hybrid conversionsa 0 2 9

Complex conversions 0 1 3

BIR 0 2 1

Totals 1 13 34

a3:1/4:0, 3:1/4:0/3:1, 1:3/0:4, or 1:3/0:4/1:3 conversion events.

58

TABLE 4. COMPARISON BETWEEN RECOMBINATION EVENTS DETECTED BY HIGH-

THROUGHPUT SEQUENCING OR BY SNP MICROARRAYSa

Type of event -ray UV

Array HTS Array HTS

Crossovers with no detectable conversion tracts 0 0 0 0

Crossovers with 3:1 or 1:3 conversion tracts 0 0 2 0

Crossovers with 4:0 or 0:4 conversion tracts 1 0 0 0

Crossovers with hybrid conversion tracts 0 1 1 1

Crossovers with complex conversion tracts 1 1 6 8

3:1 or 1:3 conversion tracts without crossovers 1 0 8 9

4:0 or 0:4 conversion tracts without crossovers 1 1 5 4

Hybrid conversion tracts without crossovers 0 1 8 7

Complex conversion tracts without crossovers 1 2 1 5

BIR 0 0 1 1

Total recombination events 5 6 32 35

aThis table includes data from two UV-induced sectored colonies and two sectored colonies induced by

gamma radiation that were analyzed by both SNP arrays and HTS. The table includes both selected

recombination events on chromosome V and unselected events on other chromosomes. All conversion

events unassociated with LOH were unselected.