Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenan...

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RESEARCH OpenAccessDifferentialgeneticinteractionsbetweenSgs1, DNA-damagecheckpointcomponentsandDNA repairfactorsinthemaintenanceofchromosome stabilityLillianDoerfler,LorenaHarris,EmilieViebranzandKristinaHSchmidt*AbstractBackground: Genomeinstabilityisassociatedwithhumancancersandchromosomebreakagesyndromes, includingBloom ssyndrome,causedbyinactivationofBLMhelicase.Numerousmutationsthatleadtogenome instabilityareknown,yethowtheyinteractgeneticallyispoorlyunderstood. Results: WeshowthatspontaneoustranslocationsthatarisebynonallelichomologousrecombinationinDNAdamage-checkpoint-defectiveyeastlackingtheBLM-relatedSgs1helicase( sgs1 mec3 )areinhibitedifcellslack Mec1/ATRkinase.Tel1/ATM,incontrast,actsasasuppressorindependentlyofMec3andSgs1.Translocationsare alsoinhibitedincellslackingDun1kinase,butnotincellsdefectiveinaparallelcheckpointbranchdefinedby Chk1kinase.Whilewehadpreviouslyshownthat RAD51 deletiondidnotinhibittranslocationformation, RAD59 deletionledtoinhibitioncomparabletothe rad52 mutation.AcandidatescreenofotherDNAmetabolicfactors identifiedExo1asastrongsuppressorofchromosomalrearrangementsinthe sgs1 mutant,becomingevenmore importantforchromosomalstabilityupon MEC3 deletion.WedeterminedthattheC-terminalthirdofExo1, harboringmismatchrepairproteinbindingsitesandphosphorylationsites,isdispensableforExo1 srolesin chromosomalrearrangementsuppression,mutationavoidanceandresistancetoDNA-damagingagents. Conclusions: OurfindingssuggestthattranslocationsbetweenrelatedgenescanformbyRad59-dependent, Rad51-independenthomologousrecombination,whichisindependentlysuppressedbySgs1,Tel1,Mec3andExo1 butpromotedbyDun1andthetelomerase-inhibitorMec1.Weproposeamodelforthefunctionalinteraction betweenmitoticrecombinationandtheDNA-damagecheckpointinthesuppressionofchromosomal rearrangementsin sgs1 cells. Keywords: genomeinstability,translocations,Sgs1,mitoticrecombination,DNA-damagecheckpointBackgroundEukaryoticcellshavemechanismsattheirdisposalfor thedetectionandrepairofspontaneousandinduced DNAlesions,thuspreventingthemfromgivingriseto potentiallyabnormaldaught ercells.However,ifthese mechanismsaredefectiveoroverwhelmedbydamage, deleteriouschromosomalrearrangementscanarise.A multitudeofgenesandgeneticpathwaysforthe maintenanceofgenomestabilityhasbeenidentified mostlyusinggeneticscreensinsimplemodelorganisms suchastheyeast Saccharomycescerevisiae .Theyinclude DNAdamagecheckpoints,DNArepairfactorsandproteinsforprocessingofreco mbinationsubstratesand intermediates[1-10].Theimportanceofthesame mechanismsformaintaininggenomestabilityinhuman cellsishighlightedbythea ssociationofmutationsin thehumanhomologuesoftheseyeastgeneswithchromosomebreakagesyndromes ,whicharecharacterized bysignsofprematureagingand/orcancerdevelopment. ThesyndromesincludeNijmegenbreakagesyndrome *Correspondence:kschmidt@usf.edu Contributedequally DepartmentofCellBiology,MicrobiologyandMolecularBiology,University ofSouthFlorida,4202E.FowlerAvenue,Tampa,FL33620,USADoerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 GENOME INTEGRITY 2011Doerfleretal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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associatedwithmutationsin NBS1 ,thehomologueof yeast XRS2 [11-13];Bloom ssyndromeandWernersyndromeassociatedwithmutationsin BLM and WRN respectively,bothrelatedtoyeast SGS1 [14,15];and ataxiatelangiectasiaassociatedwithmutationsin ATM [16],whichisrelatedtoyeast TEL1 [17]. Yeast SGS1 encodesa5 to3 DNAhelicasethatpreferentiallyunwindsthree-andfour-wayjunctionstypical ofreplicationandrecombin ationintermediatesandhas recentlybeenshowntocollaboratewithExo1inthe long-rangeprocessingofdouble-strandbreaks(DBSs) [18-21].WithoutSgs1,cellsaccumulategross-chromosomalrearrangements(GCRs),exhibitelevatedlevelsof mitoticrecombination,have areducedreplicativelifespanandaresensitivetochemicalsthatalkylateDNA orslowreplicationforks[2,22-26].AmongDNAdamagecheckpointcomponents,Mec1kinase,alsoconsideredthehomologofmammalianATR[27-29],has beenidentifiedasoneofthestrongestsuppressorsof GCRsinyeast[3,4].Othercellularphenotypesof mec1 mutantsincludeincreasedsensitivitytoDNAdamaging agentsanddeficientDNA-damagecheckpointresponse [30],instabilityofstalledforks[31],accumulationof DNAbreaks[32]and,inadditiontothesemitotic defects,deficienciesinmeioticcheckpointactivationand recombination[33-35].IncontrasttoMec1,cellslacking theTel1checkpointkinase,whichisrelatedtomammalianATM[17,36],arenotsensitivetoDNAdamaging agents[17],donotaccumulateGCRsabovewildtype levels[3],butshowtelomereerosion[36].Synergistic interactionsbetween mec1 and tel1 mutationshave beenreportedformanyphenotypes,suggestingafunctionalrelationshipandredundancybetweenthetwo kinases[3,17,37,38].Othercheckpointcomponents, suchasthoseinvolvedinsensingDNAdamage(Mec3, Rad24),appeartohaveonlysmalltomoderaterolesin suppressingGCRsinyeast[3,4].Incellslackingthe Sgs1helicase,however,Mec3andRad24stronglysuppressoverallgenomeinstab ility[3,4]aswellastheformationofspontaneous,recurringtranslocationsbetween shortidenticalsequencesinnon-allelic,butrelated, DNAsequences[10].Utilizingthehighsusceptibilityof the sgs1 mec3 mutanttorecurringtranslocationformationbetween CAN1,LYP1 and ALP1 ,wehaveinthe currentstudyconductedacandidatescreentoidentify twotypesofDNAmetabolicfactors-thosethatare requiredfortheformationofrecurringtranslocationsin the sgs1 mec3 mutantandthosethatactindependentlyofSgs1andMec3tosuppresstranslocations.For thispurpose, mec1 ,tel1 ,dun1 ,chk1 and rad59 mutationswereintroducedintothe sgs1 mec3 mutantandtheaccumulationofrecurringtranslocations wasassessed.Wefurtherdeterminedhowthelackof otherDNAmetabolicfactors( yen1 ,lig4 ,exo1 rad1 ,pol32 )affectstheaccumulationofgenome rearrangements,identifyingastrongsynergisticinteractionbetween sgs1 and exo1 .W eproposeanintegratedmodelforindependent,functionalinteractions betweenSgs1,HRsubpathwaysandvariousDNAdamage-checkpointbranchesinthesuppressionofchromosomalrearrangements.ResultsanddiscussionFunctionalinteractionbetweenSgs1andDNA-damage checkpointcomponentsMec3,Mec1,Tel1,Dun1and Chk1inthesuppressionofchromosomaltranslocationsChromosomaltranslocationsbetweenshortstretchesof homologyinnonallelicsequencesthatarenaturallypresentintheyeastgenome,suchasthehighlysimilar,but diverged CAN1 (onchromosomeV), ALP1 and LYP1 genes(onchromosomeXIV,60-65%identity),arenormallysuppressedinyeast.However,theyarerecurrentin sgs1 mutantswithcertainadditionalDNA-metabolic defects,including mec3 ,rad24 ,cac1 ,asf1 and rfc51 [10].Oneofthemutantsmostsusceptibletorecurring translocationsbetweenthe CAN1,LYP1 and ALP1 lociis the sgs1 mec3 mutant,whereastranslocationsarenot foundinthe sgs1 mec1 mutant[10].Here,wewanted totestwhetherthelackof CAN1/LYP1/ALP1 translocationsinthe sgs1 mec1 mutantmeantthatMec1was notasuppressoroftranslocationsandthereforeitsdeletionhadnoaffectontranslocationformation,orthat Mec1wasactuallyrequiredfortheformationofviable chromosomaltranslocations.Ifthelatterwastrue,we expectedthatintroducinga mec1 mutationintothe highlysusceptible sgs1 mec3 strainshouldinhibitthe accumulation CAN1/LYP1/ALP1 translocations.Indeed, wefoundthatwhiledeleting MEC1 ledtoasynergistic increase(~7-fold)intherateofallGCRtypescompared tothe sgs1 mec3 mutant(P<0.0001),screeningof GCRclonesobtainedfrom431individual sgs1 mec3 mec1 culturesfailedtorevealasingle CAN1/LYP1/ ALP1 translocation,signifyinga>7-folddecreaseinthe translocationratecomparedtothe sgs1 mec3 mutant (Table1).ThesynergisticGCRrateincreaseinthe sgs1 mec3 mec1 mutantshowsthatMec1canactivateits targetsthroughMec3-independentsensingofDNA damage.ThismayoccurbyMec1-Ddc2itselfrecognizing an dbindingtoDNAlesions[39,40]orthroughDNAdamagesensorsotherthanth eMec3clampsignalingto Mec1.ThesynergisticGCRrateincreaseinthe sgs1 mec3 mec1 mutantalsoindicatesthatthefailureto form CAN1/LYP1/ALP1 translocationswhen MEC1 is deletedisnotduetoaninabilitytoformviableGCRs, butrathersuggeststhatDNAlesionsarechanneledinto GCRpathwaysotherthanhomology-driventranslocation.Mostlikely,Mec1promoteschromosomaltranslocationsbyinhibiting denovo telomeresynthesisatDoerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page2of13

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chromosomebreaks[1],forexamplebyphosphorylating thetelomerase-inhibitorPif1[41]andbyphosphorylating Cdc13andthuspreventingitsaccumulationatDNA breaks[42].Inahaploidwildt ypecell,thesechromosomaltranslocationsareexpectedtoberaredueto restraintsplacedonhomologousrecombinationevents bytheneedforrelativelongregionsofsequenceidentity. However,whentherestraintsonhomologousrecombinationarerelaxedandspontaneousDNAlesionsarenot properlydetectedbytheDNA-damagecheckpoint,as couldbeassumedforthe sgs1 mec3 mutant,chromosomaltranslocationsarepromotedandoccurbetween muchshorterregionsofsequenceidentity,suchasthe541-bpsegmentspresentin CAN1, LYP1 and ALP1 Deleting TEL1,whichencodesanotherDNA-damage checkpointkinasethatisconsideredatleastpartially functionallyredundantwithMec1,hadthesameeffect asdeleting MEC1 ontheaccumulationofalltypesof GCR(Table1),asevidencedbythe44-foldincreasein theoverallGCRratecomparedtothesgs1 mec3 mutant(5.710-6versus1.310-7,P<0.0001).However,deleting TEL1 hadtheoppositeeffecton CAN1/ LYP1/ALP1 translocationformation(Table1).Insteadof inhibiting CAN1/LYP1/ALP1 translocationslikethe mec1 mutation,the tel1 mutationledtoanincrease (~15-fold)in CAN1/LYP1/ALP1 translocations(Table 1).Unlike mec1 mutants,mutantslackingTel1are impairedintheirabilitytomaintaintelomeres[36]and maythusbeunabletohealDNAbreaksby denovo telomereaddition.Thus,intheabsenceofTel1,DNA breaksmaybechanneledintoalternativepathwaysfor repair,suchasHR,andmorefrequentlygiveriseto CAN1 / LYP1 / ALP1 rearrangementsunderconditions thatfavoraberrantHRsuchasthoseinthe sgs1 Table1FunctionalinteractionbetweenSgs1andcomponentsoftheDNA-damagecheckpointinthesuppressionof GCRsandtranslocationsbetween CAN1,LYP1 and ALP1 genes.AllGCRtypesaCAN1 / LYP1 / ALP1 translocationsbFrequencyof CAN1/LYP1/ALP1 translocationtypescRelevantGenotypedRate95%CI Rate Frequency CAN1-ALP1CAN1-LYP1CAN1-LYP1-ALP1 wildtype 1.1<1-6.2 ND ND ND ND ND sgs1 220144-276 <7.3 0/300/300/30 0/30 rad17 5726-74 ND ND ND ND ND mec3 4618-75 <1.5 0/300/300/30 0/30 mec3rad17 4932-64 ND ND ND ND ND sgs1rad17 2515903-4160 <101 0/250/250/25 0/25 sgs1mec3 12971120-2030 173 20/1507/1503/150 7/150 sgs1mec3rad17 16901247-2230 75 2/451/451/45 0/45 tel1 2N DN DN DN DN DN D tel1mec3 453340-638 15 1/301/300/30 0/30 tel1rad17 12973-246 <8.6 0/150/150/15 0/15 sgs1tel1 22746-418 NDbND ND ND ND sgs1tel1rad17 2760022430-39653 4600 6/361/361/36 4/36 sgs1tel1mec3 5737047157-76301 2674 11/2360/2366/236 4/236 sgs1tel1mec3rad17 3196023400-51800 ND ND ND ND ND mec1 471209-859 ND ND ND ND ND sgs1mec1 1930960-2452 <10 0/1900/1900/190 0/190 sgs1mec1mec3 96285870-12100 <22 0/4310/4310/431 0/431 chk1 4225-132 ND ND ND ND ND sgs1chk1 446337-528 <15 0/300/300/30 0/30 sgs1chk1mec3 1099725-1613 147 4/301/300/30 3/30 dun1 25286-472 ND ND ND ND ND sgs1dun1 1145698-1910 <23 0/500/500/50 0/50 sgs1dun1mec3 28002270-3570 <21 0/1350/1350/135 0/135aGCRrate(Canr5-FOAr0-10).95%confidenceintervals(CIs)formedianGCRrateswerecalculatedaccordingtoNair[80],wherenon-overlappingconfidence intervalsindicatestatisticallysignificantdifferencesbetweenmedianGCRrates.GCRratesofwildtype[81], sgs1 [82], mec3,sgs1mec3 [60], tel1 [3]werereported previously.bRateofaccumulatingtranslocationsbetween CAN1,LYP1 and/or ALP1 genes(x10-10).GCRclonesfrom sgs1,mec3,sgs1mec3,sgs1tel1 and sgs1mec1 were previouslyscreenedfor CAN1 /LYP1 / ALP1 translocations[10,60].cTypesof CAN1 /LYP1 /ALP1 translocationsweredeterminedbysequencing.Ofthe20 CAN1 / LYP1 /ALP1 translocationsidentifiedamong150GCRclonesfromthe sgs1mec3 mutant,17wereidentifiedasbeingeither C/A C/L/A or C/L translocationsand3cloneshadamixtureofmultipletranslocations[60].dAllmutantswitha mec1 deletionalsocontaina sml1 deletion.Doerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page3of13

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mec3 mutant.ThatfailuretoactivateeitherTel1or Mec3-checkpointpathwayscontributesindependentlyto recurrent CAN1/LYP1/ALP1 translocationformation suggeststhatbothssDNAoverhangsorgaps,thoughtto besensedinaMec3-dependentmanner,andDSBs, thoughttobesensedinaTel1-dependentmanner,can leadto CAN1/LYP1/ALP1 translocationsandthatthey accumulateinunperturbed sgs1 cellsspontaneously. Thesynergisticincreaseinoverallgenomeinstabilityin the sgs1 mec3 tel1 mutantmightalsoindicatethat intheabsenceoflesionbindingbytheMec3clamp somelesionsarefurtherprocessedandeventually detectedbytheTel1-dependentpathway.Forexample,a stalledreplicationforkmighteventuallybeprocessed intodouble-strandedendsinanattemptatforkrestart byforkregressionortemplate-switching. Thus,bothTel1andMec1actindependentlyofMec3 andSgs1tostronglysuppressoverallgenomeinstability, buttheyaffect CAN1/LYP1/ALP1 translocationformationinoppositeways.Theinhibitionof CAN1 / LYP1 / ALP1 translocationsupon MEC1 deletionasopposedto theirincreaseupon TEL1 deletioncanmostlikelybe explainedbytheiroppositeeffectsontelomeresynthesis,withMec1inhibitingitandTel1promotingit.This isalsoconsistentwiththepreviousreportofdifferent GCRspectrainthe tel1 and mec1 singlemutants[1]. Apartfromregulatingtelomeremaintenancefactors,it isalsoconceivablethattheDNA-damagecheckpointdependentphosphorylationofhomologousrecombinationfactors,suchasRad55,Slx4andMus81[43-47] contributestodifferentialr egulationoftranslocation formationinthe sgs1 mec3 mutant. TheopposingeffectsofTel1andMec1on CAN1/ LYP1/ALP1 translocationformationledustoinvestigate otherDNA-damagecheckpointcomponentsin sgs1 and sgs1 mec3 mutants.Wefoundthatdeletionof either CHK1 or DUN1 ledtoasynergisticincreasein overallgenomeinstabilitywhencombinedwithan sgs1 mutation(P<0.0001),howeveronlythe dun1 mutationcausedafurthersignificantGCRrateincreasein the sgs1 mec3 mutant(P<0.0001,Table1)whereas the chk1 mutationdidnot(P=0.1615,Table1).AnalysisoftheGCRtypesrevealedaccumulationof CAN1 / LYP1 / ALP1 translocationsintheChk1-deficient sgs1 mec3 mu tantatasimilarrateasinthe sgs1 mec3 mutant,butnotintheDun1-deficient sgs1 mec3 mutant(Table1),indicatingthatDun1,likeMec1,promotestranslocationeventsbetween CAN1,LYP1 and ALP1 whereasChk1doesnot.Thisislikelydueto Mec1-mediatedactivationofDun1kinase,whichinturn inactivatesthetranscription repressorCrt1,thusallowingtranscriptionofseveralDNA-damageinducible genes[48,49].Chk1kinaseisalsoactivatedthrough Mec1inresponsetoDNAdamageandcausesa transientG2/Marrestbyblockinganaphaseprogression [50,51].However,incontrasttoDun1,Chk1isnot thoughttoregulateDNArepairpathways,anditsdeletiondidnotinhibittranslocationformationinthe sgs1 mec3 mutant(Table1).Asexpected,deletionof RAD17 ,whichencodesanothersubunitoftheMec3/ Rad17/Ddc1checkpointcla mp,hadasimilareffecton CAN1 / LYP1 / ALP1 translocationformationinthe sgs1 tel1 mutantasdeletionof MEC3 (Table1).Thedetectionofa CAN1 / LYP1 / ALP1 translocationintwostrains thatexpressedwildtypeSgs1( mec3 tel1 (Table1) and mec3 tel1 rad17 ( notshown))suggeststhat eveninthepresenceofwildtypeSgs1cellsmayaccumulate CAN1 / LYP1 / ALP1 translocationsaslongastheyare deficientinatleasttwoindependentsuppressorsof translocationformation,suchasTel1andMec3identifiedhere.Deletionof RAD59 inhibitsspontaneous interchromosomaltranslocationsbetweenshortrepeatsWepreviouslyshowedthat translocationsbetween CAN1 LYP1 and ALP1 inthe sgs1 mec3 mutantare Rad52-dependent,buttranslocationsstillformedwhen Rad51wasabsent[10].ToassesstheroleofotherHR factorsintranslocationformationwedeleted RAD59 in thehighlysusceptible sgs1 mec3 mutantandmeasuredtherateofaccumulatingalltypesofGCRsaswell as CAN1/LYP1/ALP1 translocations.One CAN1/ LYP1 / ALP1 rearrangementwasidentifiedamongGCRclones obtainedfrom158independentculturesofthe sgs1 mec3 rad59 mutant(Table2),indicatinga10-fold reductioninthe CAN1 / LYP1 / ALP1 translocationrate comparedtothe sgs1 mec3 mutant.Thus,similarto Rad52,Rad59isrequiredforinterchromosomaltranslocationsbetweenshortidenticalsequencesinrelated genes.IfRad59wasindeedrequiredfortranslocation formation,wepredictedthattheformationof CAN1 / LYP1 / ALP1 translocationsinthe sgs1 mec3 rad51 mutantwouldalsobeinhibitedbya rad59 mutation. Thus,wegeneratedan sgs1 mec3 rad51 rad59 mutantandscreenedfor CAN1/ LYP1 / ALP1 translocations.Among168independentGCRclonesweidentifiedone CAN1/ LYP1 / ALP1 translocation,indicativeofa 28-foldreductionofthetranslocationratecomparedto the sgs1 mec3 rad51 mutant(Table2).Thustranslocationsbetween CA N1 LYP1 and/or ALP1 canform throughRad52/Rad59-mediatedHRthatdoesnot requireRad51.Rad59hasrecentlyalsobeenshownto contributetoGCRsmediatedbycertainTy-elements andtotranslocationsinvolvingshortDNAsequencesof limitedsequenceidentity[6,52]. WhileRad52isrequiredforallHRinyeast,some DNAbreakscanberepairedbyHRpathwaysthatdo notrequireRad51,includingsingle-strandannealingDoerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page4of13

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(SSA),break-inducedreplication(BIR)andrecombination-mediatedtelomere-lengtheningTypeII[53-58]. SSAisamechanismfortherepairofaDSBbetween repeatedDNAelementsandrequiresRad59,butnot Rad51[59].Inorderfortheinterchromosomal CAN1 / LYP1 / ALP1 rearrangementstoarisebySSA,however,at leasttwoDSBswouldhavetooccurinthesamecelloneDSBwithinordownstreamof CAN1 onchromosomeVandoneDSBnear ALP1 (or LYP1 )onchromosomeXIV.Resectionwouldexposetheshortstretches ofhomologysharedby CAN1 and ALP1 (or LYP1 )[60], allowingthemtoanneal,followedbyremovalofthe nonhomologousoverhangsandligation.Rad59-dependent,Rad51-independentinterchromosomaltranslocationbetween his3 fragmentswasrecentlyshownafter inductionofHO-breaksinthetworecombiningchromosomes[61].SuchaninterchromosomalSSAevent couldalsoproducethetypesofrearrangementswehave observedbetween CAN1 LYP1 and ALP1 ;however,the endsofchromosomesVandXIVnotengagedinthe SSAeventwouldbeleftunrepairedandmostlikely wouldbelostaftercelldivi sionunlesstherecombinationeventoccursinG2/Mwhensister-chromatidsare present.Moreover,sincewehaveshownthatwildtype copiesof LYP1 and ALP1 arestillpresentinrecombinantswith CAN1/LYP1/ALP1 rearrangements,indicative ofanonreciprocaltranslocationevent[60],andthe partsofchromosomeXIVthatwouldbelostafterSSA containessentialgenes,SSAisunlikelytobethemain recombinationmechanismthatgivesriseto CAN1 / LYP1 / ALP1 rearrangements. BesidesSSA,BIRalsomatchesthegeneticrequirementsfor CAN1 / LYP1 / ALP1 translocationformation. BIRisinitiatedbyinvasionofaduplexbyasinglestranded3 endofaone-sidedDBSfollowedbyreplicationtothechromosomeend.AlthoughSgs1hasroles inrecombination,specificallysister-chromatidexchange andresolutionofrecombinationintermediates [2,9,62-64],itisnotrequiredforRad51-independent BIR[57].IncontrasttoSSA,thenonreciprocalnature ofBIReventswouldmaintainanintactcopyofchromosomeXIVinadditiontothechromosomeV/XIVtranslocation,suggestingthatitisthemorelikelymechanism involvedin CAN1/LYP1/ALP1 translocation.BIRhas beenimplicatedintherepairofone-sidedDSBs,suchas replicationforksthatcollapsedatasingle-strandbreak. BIRisalsothoughttoallowt elomerase-deficientcells ( tlc1 ),whosetelomereshaveshortenedtoapoint wherecellscannolongerproliferate,tosurviveby extendingwhatcouldbeconsideredaone-sidedDSB. Survivorscanariseeitherbyaddingsubtelomeric Y elementsinaRad51-dependentmechanism(TypeI)orby addingtelomeric(G1-3T)nrepeatsinaRad51-independent,butRad59-dependentmechanism(TypeII) [53-55].ThedifferentialrequirementforRad51and Rad59inthesetwopathwaysisthoughttoresultfrom thedifferencesinlengthandsequenceidentityofthe recombinationsubstratesforTypeIandTypeII[53]. Thelong,nearlyidentical(~1%variationwithinthe samestrain) Y elements[65]arethoughttobebetter substratesforRad51-mediatedstrandinvasion,whereas Rad59isabletousetheshorterstretchesofhomology likelytobefoundwithinthehighlyvariable(G1-3T)nrepeats[53].BesidesBIR,evidenceofhomology-length dependencyisalsoseeningeneconversion,withRad59 becomingincreasinglyimportantasthelengthof sequencehomologydecreases [59].Thislength-dependencymayalsoexplainourobservationthat CAN1 / LYP1 / ALP1 rearrangements,whichshowshortregions ofhomologyatthebreakpoints[10,60],areinhibitedby deletionof RAD59,butnotbydeletionof RAD51 Despitethisdifferentiale ffectonchromosomerearrangementsbetween CAN1 LYP1 and ALP1 ,weobserved nodifferenceintherateofoverallgenomeinstability Table2Effectofhomologousrecombinationmutations ontheabilityofthe sgs1mec3 mutanttoaccumulate GCRsandformrearrangementsbetweenthe CAN1,LYP1 and ALP1 genes.AllGCR typesaCAN1 / LYP1 / ALP1 translocationsbRelevantgenotypeRate95%CIRateFrequency wildtype1.1<1-6.2NDND rad51a<8<7-15NDND rad52 13816-267NDND rad59 2413-50NDND sgs1a220144-276<7.30/30 mec3a4618-75<1.50/30 sgs1rad59 126107-300NDND sgs1rad59rad51 11849-154NDND sgs1mec3a12971120-203017320/150 sgs1mec3rad51a1491NDc1984/30 sgs1mec3rad52a3168NDc<230/136 sgs1mec3rad59 24761595-3187161/158 sgs1mec3rad59rad51 1124734-146071/168aMedianrateofcellsresistanttocanavanineand5-FOA(Canr5-FOAr0-10). 95%confidenceintervals(CIs)formedianGCRrateswerecalculated accordingtoNair[80],wherenon-overlappingconfidenceintervalsindicate statisticallysignificantdifferencesbetweenmedianGCRrates.GCRratesfor wildtype[81], sgs1 [82], mec3,sgs1mec3 [60], rad51,sgs1mec3rad51 and sgs1 mec3rad52 mutants[10]werereportedpreviouslyandareincludedfor comparison.bRateofaccumulatingtranslocationsbetween CAN1,LYP1 and/or ALP1 (Canr5-FOAr0-10).GCRclonesfrom sgs1,mec3,sgs1mec3,sgs1mec3rad51,sgs1 mec3rad52 werepreviouslyscreenedfor CAN1/LYP1/ALP1 translocations [10,60].ND,notdetermined.cTodetermine95%CIsfor sgs1mec3rad51 and sgs1mec3rad52 mutants, GCRrateswerere-measuredforthecurrentstudy.TheGCRrateforthe sgs1 mec3rad51 mutantwas193310-10(95%CIs:601-224010-10)andtheGCR rateforthe sgs1mec3rad52 mutantwas222010-10(951-347010-10).The previouslyreportedratesfallwithinthe95%CIsdeterminedinthecurrent study.Doerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page5of13

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between sgs1 mec3 rad51 and sgs1 mec3 rad59 mutants(P=0.6892,Table2),suggestingthattheDNA lesionsthatgiverisetoviableGCRsareaccessibleto multiplerepairpathways.Candidatescreenreveals EXO1 asastrongsuppressorof GCRformationincellslackingSgs1ToassessthepossibleroleofotherDNAmetabolicfactorsinthesuppressionorformationofGCRsincells lackingSgs1,weintroduced exo1 pol32 ,rad1 ,lig4 and yen1 mutationsinto sgs1 and sgs1 mec3 mutants.Screeningofthesingle,doubleandtriple mutantsrevealedthat RAD1,POL32,LIG4 and YEN1 arenotstrongsuppressorsofGCRsinwildtypecells,or in sgs1 or sgs1 mec3 mutants(Table3).However, whenweassessedtheformationof CAN1/LYP1/ALP1 translocationsin sgs1 mec3 mutantswith pol32 or rad1 mutationswefoundthatinbothtriplemutants CAN1/LYP1/ALP1 translocationswereinhibited,revealingone CAN1 / LYP1 translocationamong98 independentGCRclonesinthe sgs1 mec3 pol32 mutantandnone(0/55)inthe sgs1 mec3 rad1 mutant.Pol32,anonessent ialsubunitofpolymerase thatpromotesprocessivityofthepolymerase,isnot requiredforSSA,butforDNArepairprocessesthat involveextensiveDNAsynthesis,suchasBIR[66],consistentwithBIRbeingapathwayfor CAN1 / LYP1 / ALP1 translocationformation.AlthoughRad1,asubunitof theRad1-Rad10nucleasecriticalfortheremovalofnonhomologousoverhangsfromannealedsinglestrandsin processessuchasSSA[67,68],hasnotbeenshownto berequiredforBIR,ithasbeenimplicatedinthe removalofnonhomologousoverhangsduringGCRformation[69]andinrecombinationeventsthatcombine BIRandSSAprocesses[70,71]. Deletionof EXO1 ,codingforanucleasewith5 to3 exonucleaseandflap-endonucleaseactivities,whichhas rolesinmitoticandmeioticrecombinationaswellas mutationavoidanceandisthoughttocooperatewith Sgs1intheprocessingofDSBs[19,72],inducedthelargestsynergisticGCRrateincreasewehaveobservedto dateinthe sgs1 mutant.While sgs1 and exo1 single mutantsexhibitedmoderatelyincreasedGCRratescomparedtowildtype,theGCRrateofthe sgs1 exo1 mutant wasseveralhundred-foldhigherthantherates ofthesinglemutants(P<0.0001,Table3).ThisGCR rateincreasedanother26-foldupondeletionof MEC3 (P<0.0001,Table3).ScreeningofGCRsobtainedfrom 66independentculturesofthe sgs1 mec3 exo1 mutantidentifiedtwo CAN1 / LYP1 / ALP1 translocations, indicatinga~200-foldincreaseinthe CAN1 / LYP1 / ALP1 translocationratecomparedtothe sgs1 mec3 mutant(3.510-6versus1.710-8). Exo1containsconservedN-terminalN-andI-nucleasedomains,apparentlyseparatedbyashortdisordered linker,andbindingsitesforthemismatchrepair(MMR) proteinsMlh1andMsh2havebeenlocatedwithinthe C-terminalhalfofExo1[72-74],whichispredictedto beintrinsicallydisordered(Figure1A).Fourphosphorylationsites(S372,S567,S587,S692)requiredforthe regulationoftheDNA-damageresponsehavealsobeen locatedinthedisorderedC-terminus[75].Todetermine iftheC-terminusofExo1playsaroleinthesuppression ofgenomeinstabilityinthe sgs1 mutantweconstructedasetofC-terminaldeletionsrangingfrom100 to400residues(Figure1Aand1B).Wefoundthatthe C-terminal260residuesofExo1,makingup37%ofthe protein,playnomajorroleinsuppressingtheaccumulationofGCRsinthe sgs1 mutant(Table4).Totestthe possibilitythattheC-terminuswithitsbindingsitesfor MMRproteinsmightberequiredforExo1 srolein mutationavoidance,butnotforitsroleinsuppressing GCRs,weutilizedafluctuationassaytodeterminethe rateofaccumulatingcanavanineresistance(Canr) Table3Effectof lig4 ,exo1 ,rad1 ,pol32 and yen1 mutationsontheaccumulationofGCRsincheckpointproficientandcheckpoint-deficient sgs1 mutantsRelevantgenotypeaGCRrateb95%CIcwildtype 1.1 <1-6.2 exo1 24 7-79 sgs1 220 144-276 sgs1mec3 1297 1120-2030 exo1sgs1 4380030400-186000 exo1mec3 30 12-39 exo1mec3sgs1 1168498549530-3251000 sgs1mec3exo1lig4 895988701149-1236740 lig4 16 ND sgs1lig4 80 35-254 sgs1mec3lig4 1335 948-2140 yen1 <5 <4-6 sgs1yen1 81 57-265 sgs1mec3yen1 1089 254-2540 pol32 20 15-26 sgs1pol32 25 <24-105 sgs1mec3pol32 2317 1800-3110 rad1 10 <9-23 sgs1rad1 63 25-356 sgs1mec3rad1 1173 1020-1540aStrainswithmultiplegenedeletionswereconstructedbysporulationofthe appropriateheterozygousdiploids.GCRrateswith95%confidenceintervals (CIs)forwildtype[81], sgs1 [82], sgs1mec3 [60]and lig4 [1]werereported previouslyandareincludedforcomparison.Sporeswithboth sgs1 and pol32 mutationsgrewveryslowlyandexhibitedalowviablecellcounton YPDintheGCRassay.bTherateofaccumulatinggross-chromosomalrearrangements(GCRs)is calculatedbyselectingforcellsresistanttocanavanine(Canr)and5-fluorooroticacid(5-FOAr)andisexpressedasCanr5-FOAr0-10[77].c95%confidenceintervals(CI)formedianGCRrateswerecalculated accordingtoNair[80].Doerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page6of13

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mutationsinstrainsexpressingthevariousC-terminal Exo1truncations(Table5).AsintheGCRassay,deletionofupto260residueshadnoeffectontheCanrmutationrate(P=0.3524)whereasdeletionof280or moreresiduescausedanullphenotype(P=0.0001). Similarly,onlydeletionof280ormoreresiduescaused sensitivitytomethylmethanesulfonate(MMS)(Figure 1C).Nosensitivityto200mMhydroxyureawas observedforanyofthe exo1 mutants(Figure1C).Thus, deletionofupto260residuescausedaphenotype 190 125 80 wildtype exo1 exo1C100.myc exo1C200.myc exo1C240.myc exo1C260.myc exo1C280.myc exo1C300.myc exo1C400.myc YPD 0.05% MMS 200 mM HU B C A Exo1 (702 residues) 0 0.5 1 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 Figure1 ExpressionofC-terminaltruncationsofExo1andsensitivitytoDNA-damagingagents (A) IntrinsicdisorderpredictionofExo1 usingtheIUPredalgorithminwhichvaluesabove0.5indicateresiduespredictedtobeintrinsicallydisorderedandvaluesbelow0.5tobe ordered.TheN-terminus,harboringconservedN-andI-nucleasedomains,ispredictedtobeordered,whereastheC-terminus,whichappears devoidofenzymaticactivitybutcontainsphosphorylationsitesandsitesforinteractionwithmismatchrepairproteins,isdisordered.Thesitesat whichtheExo1truncationsexaminedinthisstudyterminateareindictedbyverticaldottedlines.Thelocationofconserveddomainswas adaptedfromreference[71]:nucleasedomains(orangeboxes,16-96aa,123-257aa),Mlh1interactiondomain(greenbox,400-702aa)andthe Msh2interactiondomain(bluebox,368-702aa).PhosphorylationsitesatS372,S567,S587andS692,implicatedincheckpointregulation[74],are indicatedbyredasterisks. (B) Westernblotanalysisofexpressionofmyc-epitopetaggedexo1truncationsandwildtypeExo1.Molecularweight markers(kD)areindicatedontheleft. (C) CellsexpressingExo1truncationslacking280ormoreC-terminalresiduesareassensitiveto0.05% MMSasthe exo1 mutantwhereascellsexpressingexo1truncationslacking260orfewerC-terminalresiduesshowwildtypelevelsofresistance toMMS.Nosensitivityto200mMhydroxyureawasobservedforanyofthetestedyeaststrains. Doerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page7of13

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similartowildtypeinallassaystestedhere,whereas deletionof280ormoreresiduescausedanull( exo1 ) phenotype. InadditiontoprovidingMMRproteininteraction sites,theC-terminusofExo1containsfourphosphorylationsites(S372,S567,S587,S692),whichwererecently showntobeimportantfortheregulationoftheDNA damagecheckpointinresponsetouncappedtelomeres ina cdc13-1 mutant[75].Unlikeina cdc13-1 mutant, wedidnotdetectExo1phosphorylationinthe sgs1 mutant(datanotshown),anddeletionoftheC-terminal thirdofExo1( exo1C260 ),whichcontainsthreeofthe fourphosphorylationsites(S567,S587,S692),hadno effectonExo1functionintheassaysusedhere(Canrmutationrate,GCRassay,MMSsensitivity).Thefourth phosphorylationsite(S372)ispresentinboththe exo1C260 mutantandthe exo1C280 mutantand,therefore,isnotresponsiblefor thedifferentphenotypes associatedwiththetwoalleles.Thus,theknownphosphorylationsitesinExo1donotappeartoberequired forExo1 sroleinmutationavoidance,resistanceto MMSorsuppressionofGCRsina sgs1 mutant. Instead,itislikelythatthe C280 deletionaffectsExo1 nucleaseactivitydirectlybydisruptingintramolecular interactionswiththeN-terminus.Thelossofyet unknownposttranslationalmodificationsinthissegment ofExo1oranindirecteffectcausedbythelossofinteractionwithothercellularfactorscouldalsoleadtothe deficiencyofthe exo1 C280 allele. Besidestheoverallincreaseingenomeinstability, CAN1/ LYP1 / ALP1 rearrangementsseeninthe sgs1 mec3 mutantwerealsopresentinthe sgs1 mec3 exo1 mutant.Normally,Exo1andSgs1functionin independentendresectionpathwaysthatcooperatein theprocessingofDSBs,especiallythelong-rangeresectionofthe5 -strand[19,20],andMarreroandSymington[21]recentlyshowedthatthisextensiveresection inhibitsBIRinaplasmid-basedassay.BesidesupregulationofBIR,whichwasalsoaccompaniedbychromosomerearrangements,the exo1 sgs1 mutantwasalso moreproficientin denovo telomeresynthesisatHOendonuclease-inducedchromosomebreaks[18,21].The combinationofincreasedBIRandmoreefficient de novo telomereaddition,bothofwhichhavebeenidentifiedasmajormechanismsforthehealingofchromosomeVbreaksintheGCRassay[76,77],likelyalso explainstheremarkablystrongaccumulationofgenome rearrangementsoriginatingfromspontaneousDNA lesionsinthe exo1 sgs1 mutantstudiedhere.Our studyfurtheraddsthatthe exo1 sgs1 mutanthas evengreaterpotentialfor theaccumulationofviable genomerearrangements,whichissuppressed(~26-fold) inthe sgs1 exo1 mutantbyMec3-dependentDNAdamagecheckpointfunctions(P<0.0001).Nonhomologousendjoiningdoesnotappeartobeasignificant sourceforthesegenomerearrangements,asindicated bythelackofanyeffectof LIG4 genedeletionin mutantswithvariouscombinationsof sgs1 exo1 and mec3 mutations(e.g.,GCRrateof sgs 1 mec3 exo1 comparedto sgs1 mec3 exo1 lig4 ,P=0.3953, Table3);however,itisalsoplausiblethatintheabsence ofonerepairpathwayDNAlesionssimplybecomesubstratesforvariousotheravailablerepairpathways.ConclusionOurresultsindicatethatspontaneous,interchromosomaltranslocationsbetweenshortregionsofsequence Table4Effectof exo1 mutationsontheaccumulationof GCRsinwildtypecellsorcellslackingSgs1helicase.RelevantgenotypeGCRrate (Canr5-FOAr0-10) 95%CIa(Canr5-FOAr0-10) sgs1 8957-177 exo1 24 7-79 exo1 sgs1 40484 31076-49848 EXO1.myc 5 4.4-5.3 exo1C100.myc 5 4-6 exo1C200.myc <4 <3.8-4.8 exo1C260.myc <11 <8-79 exo1C280.myc <11 <8-29 exo1C300.myc <18 <5-70 exo1C400.myc 13 5-41 sgs1 EXO1.myc 78 29-118 sgs1 exo1C100.myc 125 80-186 sgs1 exo1C200.myc 158 94-215 sgs1 exo1C260.myc 230 166-265 sgs1 exo1C280.myc 26840 22925-34036 sgs1 exo1C300.myc 31070 22871-33753 sgs1 exo1C400.myc 48190 39133-54471a95%confidenceintervals(CI)formedianGCRrateswerecalculated accordingtoNair[80]. Table5EffectofC-terminaldeletionsofExo1onthe spontaneousmutationrateatthe CAN1 locus.Relevant genotype CAN1 (Canr07) 95%CIa(Canr07) Increaseover wildtype wildtype3.272.50-5.821 exo1 11.4710.1028.52 3.5 exo1C100.myc 3.642.92-4.701.1 exo1C200.myc 5.313.90-5.901.6 exo1C260.myc 3.892.89-5.921.2 exo1C280.myc 8.376.94-16.182.6 exo1C300.myc 10.728.55-19.883.3 exo1C400.myc 13.169.06-18.194.0a95%confidenceintervals(CI)formedianCanrrateswerecalculated accordingtoNair[80].Doerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page8of13

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identity(5-41bp),suchasthosepresentinthe CAN1 LYP1 and ALP1 genesusedinourassay,arepromoted byMec1/Dun1/Rad59-depe ndentpathwayswhereas Tel1,Mec3andSgs1actasindependentsuppressors (Figure2).TherequirementforPol32andRad1inthe translocationprocessfurthersuggeststheneedfor extensiveDNAsynthesis,suchasseeninBIR,andthe removalofnonhomologousoverhangsfromannealed single-strands,criticalforSSAandimplicatedinGCR formation.Exo1nucleaseisasuppressorofoverallgenomerearrangementsaswellas CAN1 / LYP1 / ALP1 translocationswhencellslackSgs1orbothSgs1andMec3. Thatthedisordered,C-terminalthirdisdispensablefor Exo1functioninourassaysfurtherindicatesthatphysicalinteractionwithMMRproteinsinthisregionand regulationofExo1functioninresponsetoDNA-damage arenotimportantforExo1 sroleinthesuppressionof spontaneousGCRs,mutationavoidanceandresistance toMMS.MethodsYeaststrainsandmediaAllstrainsusedinthisstudyarederivedfromKHSY802 ( MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl, hom3-10,ade2 1,ade8,hxt13::URA3)ortheisogenic strainoftheoppositematingtype.DesiredgenedeletionswereintroducedbyHR-mediatedintegrationof PCRproductscontainingaselectablemarkercassette flankedby50-ntsequencescomplementarytothetarget locus[78].C-terminaltruncationsofExo1were Homology search HR intermediate Resolution Tel 1 Mec3 Mec1/Dun1 Mutagenic repair (nonC/L/A ) Sgs1 Sgs1 Sgs1 Mutagenic repair (C/L/A ) S gs1 Nonmutagenic repair Sgs1 Chk1 S G2/M Telomere synthesis (Pif1, Cdc13) Rad59 Figure2 Factorsaffectingthesuppressionandpromotionofchromosomaltranslocationsbetweenshortsegmentsofhomologyin CAN1 LYP1 and ALP1 incellslackingSgs1 .IntheabsenceofSgs1,translocationsbetween CAN1,LYP1 and ALP1 (referredtoas C/L/A )are independentlysuppressedbythecheckpointcomponentsMec3andTel1(showninredfont),assuggestedbythesynergisticincreasesinthe GCRrateandthe C/L/A translocationrateofthe sgs1 mutantupondeletionof MEC3 ( sgs1 mec3 )andsubsequently TEL1 ( sgs1 mec3 tel1 ). IfMec3isabsent( sgs1 mec3 ), C/L/A translocationsformthroughapathwaythatrequiresMec1,Dun1andhomologousrecombination(HR) factors(showningreenfont),especiallyRad52andRad59.Mec1mostlikelypromotestranslocationsbyinhibiting denovo telomereadditionsby regulatingPif1andCdc13.Inadditiontomutagenicrepairthatleadsto C/L/A translocations,othertypesofmutagenicrepair(e.g.,translocations betweenothergenes, denovo telomereadditions,deletions,insertions,inversions)andmostlikelyalsononmutagenicrepairproductsare formed.If,inadditiontoMec3,Tel1isalsoabsent(e.g., sgs1 mec3 tel1 ),anevengreaternumberofDNAlesionsarechanneledthroughthe Mec1-dependent, C/L/A -promotingpathway.Incontrastto dun1 ,the chk1 mutationdoesnotleadtoasignificantGCRrateincreaseinthe sgs1 mec3 mutantanddoesnotinhibit C/L/A translocationformation.Possibly,theinabilitytoregulatecellcycleprogressionintheabsenceof Chk1leadstoincreasedformationofinviableGCRs.Dottedlinesindicateeventsthatoccurintheabsenceoftheproteinfromwhichthearrow originates;fulllinesindicateeventsthatoccurinthepresenceoftheprotein. Doerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page9of13

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Table6 Saccharomycescerevisiae strainsusedinthisstudyStrainIDGenotype KHSY802 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3 RDKY3721aMATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,rad17::HIS3 RDKY3739aMATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,dun1::HIS3 RDKY3745aMATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,chk1::HIS3 RDKY5209aMATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,tel1:G418 KHSY773 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sml1::TRP1,mec1::HIS3 KHSY884 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,rad51::HIS3 KHSY906 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,mec3::HIS3 KHSY1330 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::HIS3,mec1::TRP1,sml1::G418 KHSY1498 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::HIS3,mec1::TRP1,sml1::G418,mec3::G418 KHSY1524 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1 KHSY2260 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,chk1::HIS3 KHSY2265 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,rad17::HIS3 KHSY2280 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,mec3::HIS3,rad59::G418 KHSY2283 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,dun1::HIS3 KHSY2317 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,tel1::G418,mec3::HIS3 KHSY2320 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,mec3::HIS3 KHSY2330 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,yen1::loxP-G418-loxP KHSY2331 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,lig4::loxP-G418-loxP KHSY2336 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,rad1::loxP-G418-loxP KHSY2338 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1:loxp-G418-loxp KHSY2388 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,rad59::G418 KHSY2402 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,exo1::loxP-G418-loxP KHSY2408 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1 exo1::loxP-G418-loxP,mec3::HIS3 KHSY2424 MATa,ura3-52,trp1 63, his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1 rad1::loxP-G418-loxP KHSY2434 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1 rad1::loxP-G418-loxP,mec3::HIS3 KHSY2447 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1 lig4::loxP-G418-loxP KHSY2448 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1 yen1::loxP-G418-loxP KHSY2449 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1 yen1::loxP-G418-loxP,mec3::HIS3 KHSY2559 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,mec3::G418,rad17::HIS3 KHSY2565 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,mec3::G418,rad17::HIS3 KHSY2579 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,lig4::G418,mec3::HIS3 KHSY2585 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,tel1::G418,rad17::HIS3 KHSY2588 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,tel1::G418,rad17::HIS3 KHSY2662 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,mec3::HIS3,chk1::HIS3 KHSY2665 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,mec3::HIS3,dun1::HIS3 KHSY2786 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,exo1::loxP-G418-loxP,lig4::loxP-G418-loxP, mec3::HIS3 KHSY3086 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,mec3::G418,rad17::HIS3,tel1::HIS3 KHSY3223 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,sgs1::TRP1,mec3::HIS3,tel1::G418 KHSY3231 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,rad17::H1S3,mec3::HIS3,tel1::G418 KHSY3265 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 C300.MYC.HIS KHSY3271 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 C400.MYC.HIS KHSY3274 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 C200.MYC.HIS,sgs1::TRP1 KHSY3278 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 C100.MYC.HIS,sgs1::TRP1 KHSY3282 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 C100.MYC.HIS KHSY3287 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,EXO1.MYC.HIS,sgs1::TRP1 KHSY3395 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,EXO1.MYC.HIS KHSY3396 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 200.MYC.HIS Doerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page10of13

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constructedbyreplacingthedesiredDNAsequenceat thechromosomal EXO1 locuswithamyc-epitope encodingsequenceamplifiedfrompFA-13Myc. His3MX6(agiftfromMarkLongtine,WashingtonUniversity,St.Louis).ExpressionofExo1truncationalleles wasconfirmedbyWesternb lottingusingmonoclonal anti-c-mycantibody(Covance).Allhaploidstrainswith multiplegenedeletionswereobtainedbysporulating diploidsheterozygousforthedesiredmutationstominimizetheriskofobtainingsu ppressors.Thiswasespeciallyimportantforcombinationsofmutationsknown tocausefitnessdefects,suchas sgs1 and pol32 .Spore isolationwasfollowedbygenotypingofmeioticproductsbyspottingonselectivemediaorbyPCR.All strainsusedinthisstudyarelistedinTable6.Media forpropagatingyeaststrainshavebeenpreviously described[76,77].SensitivitytoDNAdamagingagentsHUandMMSCellculturesweregrowninyeastextract/peptone/dextrose(YPD)mediaandadjustedtoOD600=1.Tenfold dilutionswerespottedonYPD,YPDsupplementedwith 0.05%methyl-methanesulfonate(MMS)andYPDsupplementedwith200mMhydroxyurea(HU).Colony growthwasdocumentedafterincubationat30Cfor3 days.FluctuationAssaysRatesofaccumulatingspont aneousgross-chromosomal rearrangements(GCRs)weredeterminedbyfluctuation analysisandthemethodofthemedianaspreviously described[77,79].CellswithGCRsweredetectedby theirresistancetocanavanineand5-fluoro-oroticacid (Canr5-FOAr)duetosimultaneousinactivationofthe CAN1 and URA3 genes,bothlocatedwithina12kb nonessentialregionontheleftarmofchromosomeV. ThemedianGCRrateisrepo rtedwith95%confidence intervals[80].GCRcloneswerescreenedbyPCRto identifycloneswithrearrangementsbetween CAN1 on chromosomeVand LYP1 and/or ALP1 (collectively referredtoas CAN1 / LYP1 / ALP1 rearrangementsinthe text),locatedinoppositeorientationsonthesamearm ofchromosomeXIV[10].Todeterminetherateof accumulatingspontaneousmutationsthatleadtoinactivationofthe CAN1 gene,3-mlYPDculturesexpressing wildtypeExo1orC-termin altruncationsofExo1were grownovernightandaliquotswereplatedonsynthetic medialackingarginine(USBiological)supplemented with240mgml-1canavanine(Sigma),andonYPDto obtaintheviablecellcount.Colonieswerecountedafter twodaysofincubationat30C.Atleasttwelveindependentculturesfromthreeisolateswereanalyzedper yeaststrain.ThemedianCanrmutationrateisreported with95%confidenceintervals [80].StatisticalsignificanceofdifferencesinGCRrateswasevaluatedby usingtheMann-WhitneytestandprogramsfromDr.R. LowryatVassarCollegehttp://faculty.vassar.edu/lowry/ VassarStats.html.ProteinextractionandWesternblotanalysisCellsweregrowninYPDuntiltheyreachedOD600= 0.5.Wholecellextractwaspreparedfrom5mlofcultureusingastandardtrichloroaceticacid(TCA) extraction.Briefly,cells werepelleted,vortexedwith glassbeadsfor10minutesin200 lof20%TCA,followedbycentrifugationfor2minutes.Thepelletwas resuspendedinsamplebufferandpHwasneutralized with2MTrisbuffer(pH7.6).Proteinswereseparated byPAGE,transferredtoaPVDFmembraneandincubatedwithmonoclonalanti-c-mycantibody(Covance) todetectmyc-taggedproteins.Bandswerevisualized usingECLPlusChemil uminescencekit(GE Healthcare).Listofabbreviations BIR:break-inducedreplication;Canr:canavanineresistant;CI:confidence interval;DSB:double-strandbreak;5-FOAr:5-fluoro-oroticacidresistant;GCR: gross-chromosomalrearrangement;HR:homologousrecombination;HU: hydroxyurea;MMR:mismatchrepair;MMS:methylmethanesulfonate;SSA: single-strandannealing;TCA:trichloroaceticacid;YPD:yeastextract/ peptone/dextrose. Table6 Saccharomycescerevisiae strainsusedinthisstudy (Continued)KHSY3402 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 300.MYC.HIS,sgs1::TRP1 KHSY3635 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1::loxP-G418-loxP,mec3::HIS3 KHSY3843 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 400.MYC.HIS,sgs1::TRP1 KHSY3849 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 280.MYC.HIS KHSY3857 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 280.MYC.HIS,sgs1::TRP1 KHSY3860 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 260.MYC.HIS KHSY3866 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 240.MYC.HIS KHSY3868 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 260.MYC.HIS KHSY3869 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1, ade8,hxt13::URA3,exo1 260.MYC.HIS,sgs1::TRP1 KHSY3875 MATa,ura3-52,trp1 63,his3 200,leu2 1,lys2Bgl,hom3-10,ade2 1,ade8,hxt13::URA3,exo1 280.MYC.HIS,sgs1::TRP1aRDKYstrainswereakindgiftfromRichardKolodner(LudwigInstituteforCancerResearch,UniversityofCalifornia-SanDiego).Doerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page11of13

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Acknowledgements WethankthemembersoftheSchmidtlabandGaryDaughdrill(University ofSouthFlorida)forhelpfuldiscussions,RichardKolodner(LudwigInstitute forCancerResearch,UniversityofCaliforniaSanDiego)forsendingstrains andMarkLongtine(WashingtonUniversity,St.Louis)forplasmids.Thiswork wassupportedbyNationalInstitutesofHealthgrant5R01GM081425toKHS. Authors contributions LDconstructedyeaststrains,performedexperiments,analyzeddataand performedstatisticalanalyses.LHconstructedyeaststrains,performed experiments,analyzeddataandperformedstatisticalanalyses.EV constructedyeaststrainsandperformedexperiments,KHS.designedthe study,analyzeddataandwrotethemanuscript.Allauthorshavereadand approvedthefinalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:14June2011Accepted:31October2011 Published:31October2011 References1.MyungK,ChenC,KolodnerRD: Multiplepathwayscooperateinthe suppressionofgenomeinstabilityinSaccharomycescerevisiae. Nature 2001, 411 :1073-1076. 2.MyungK,DattaA,ChenC,KolodnerRD: SGS1,theSaccharomyces cerevisiaehomologueofBLMandWRN,suppressesgenomeinstability andhomeologousrecombination. NatGenet 2001, 27 :113-116. 3.MyungK,DattaA,KolodnerRD: Suppressionofspontaneous chromosomalrearrangementsbySphasecheckpointfunctionsin Saccharomycescerevisiae. Cell 2001, 104 :397-408. 4.MyungK,KolodnerRD: Suppressionofgenomeinstabilitybyredundant S-phasecheckpointpathwaysinSaccharomycescerevisiae. ProcNatl AcadSciUSA 2002, 99 :4500-4507. 5.MyungK,PennaneachV,KatsES,KolodnerRD: Saccharomycescerevisiae chromatin-assemblyfactorsthatactduringDNAreplicationfunctionin themaintenanceofgenomestability. ProcNatlAcadSciUSA 2003, 100 :6640-6645. 6.PutnamCD,HayesTK,KolodnerRD: Specificpathwaysprevent duplication-mediatedgenomerearrangements. Nature 2009, 460 :984-989. 7.PutnamCD,JaehnigEJ,KolodnerRD: PerspectivesontheDNAdamage andreplicationcheckpointresponsesinSaccharomycescerevisiae. DNA Repair(Amst) 2009, 8 :974-982. 8.PutnamCD,PennaneachV,KolodnerRD: Saccharomycescerevisiaeasa modelsystemtodefinethechromosomalinstabilityphenotype. MolCell Biol 2005, 25 :7226-7238. 9.SchmidtKH,KolodnerRD: Suppressionofspontaneousgenome rearrangementsinyeastDNAhelicasemutants. ProcNatlAcadSciUSA 2006, 103 :18196-18201. 10.SchmidtKH,WuJ,KolodnerRD: ControlofTranslocationsbetweenHighly DivergedGenesbySgs1,theSaccharomycescerevisiaeHomologofthe Bloom sSyndromeProtein. MolCellBiol 2006, 26 :5406-5420. 11.AntocciaA,KobayashiJ,TauchiH,MatsuuraS,KomatsuK: Nijmegen breakagesyndromeandfunctionsoftheresponsibleprotein,NBS1. GenomeDyn 2006, 1 :191-205. 12.CarneyJP,MaserRS,OlivaresH,DavisEM,LeBeauM,YatesJR,HaysL, MorganWF,PetriniJH: ThehMre11/hRad50proteincomplexand Nijmegenbreakagesyndrome:linkageofdouble-strandbreakrepairto thecellularDNAdamageresponse. Cell 1998, 93 :477-486. 13.VaronR,VissingaC,PlatzerM,CerosalettiKM,ChrzanowskaKH,SaarK, BeckmannG,SeemanovaE,CooperPR,NowakNJ, etal : Nibrin,anovel DNAdouble-strandbreakrepairprotein,ismutatedinNijmegen breakagesyndrome. Cell 1998, 93 :467-476. 14. EllisNA,GrodenJ,YeTZ,StraughenJ,LennonDJ,CiocciS,ProytchevaM, GermanJ: TheBloom ssyndromegeneproductishomologoustoRecQ helicases. Cell 1995, 83 :655-666. 15.YuCE,OshimaJ,FuYH,WijsmanEM,HisamaF,AlischR,MatthewsS, NakuraJ,MikiT,OuaisS, etal : PositionalcloningoftheWerner s syndromegene. Science 1996, 272 :258-262. 16.SavitskyK,Bar-ShiraA,GiladS,RotmanG,ZivY,VanagaiteL,TagleDA, SmithS,UzielT,SfezS, etal : Asingleataxiatelangiectasiagenewitha productsimilartoPI-3kinase. Science 1995, 268 :1749-1753. 17.MorrowDM,TagleDA,ShilohY,CollinsFS,HieterP: TEL1,anS.cerevisiae homologofthehumangenemutatedinataxiatelangiectasia,is functionallyrelatedtotheyeastcheckpointgeneMEC1. Cell 1995, 82 :831-840. 18.LydeardJR,Lipkin-MooreZ,JainS,EapenVV,HaberJE: Sgs1andexo1 redundantlyinhibitbreak-inducedreplicationanddenovotelomere additionatbrokenchromosomeends. PLoSGenet 2010, 6 :e1000973. 19.MimitouEP,SymingtonLS: Sae2,Exo1andSgs1collaborateinDNA double-strandbreakprocessing. Nature 2008, 455 :770-774. 20.ZhuZ,ChungWH,ShimEY,LeeSE,IraG: Sgs1helicaseandtwo nucleasesDna2andExo1resectDNAdouble-strandbreakends. Cell 2008, 134 :981-994. 21.MarreroVA,SymingtonLS: ExtensiveDNAendprocessingbyexo1and sgs1inhibitsbreak-inducedreplication. PLoSGenet 2010, 6 :e1001007. 22.CobbJA,BjergbaekL,GasserSM: RecQhelicases:attheheartofgenetic stability. FEBSLett 2002, 529 :43-48. 23.FreiC,GasserSM: TheyeastSgs1phelicaseactsupstreamofRad53pin theDNAreplicationcheckpointandcolocalizeswithRad53pinS-phasespecificfoci. GenesDev 2000, 14 :81-96. 24.IraG,MalkovaA,LiberiG,FoianiM,HaberJE: Srs2andSgs1-Top3 suppresscrossoversduringdouble-strandbreakrepairinyeast. Cell 2003, 115 :401-411. 25.LeeSK,JohnsonRE,YuSL,PrakashL,PrakashS: Requirementofyeast SGS1andSRS2genesforreplicationandtranscription. Science 1999, 286 :2339-2342. 26.VersiniG,CometI,WuM,HoopesL,SchwobE,PaseroP: TheyeastSgs1 helicaseisdifferentiallyrequiredforgenomicandribosomalDNA replication. EmboJ 2003, 22 :1939-1949. 27. BentleyNJ,HoltzmanDA,FlaggsG,KeeganKS,DeMaggioA,FordJC, HoekstraM,CarrAM: TheSchizosaccharomycespomberad3checkpoint gene. EmboJ 1996, 15 :6641-6651. 28.CarrAM: ControlofcellcyclearrestbytheMec1sc/Rad3spDNA structurecheckpointpathway. CurrOpinGenetDev 1997, 7 :93-98. 29.CimprichKA,ShinTB,KeithCT,SchreiberSL: cDNAcloningandgene mappingofacandidatehumancellcyclecheckpointprotein. ProcNatl AcadSciUSA 1996, 93 :2850-2855. 30.WeinertTA,KiserGL,HartwellLH: Mitoticcheckpointgenesinbudding yeastandthedependenceofmitosisonDNAreplicationandrepair. GenesDev 1994, 8 :652-665. 31.TerceroJA,DiffleyJF: RegulationofDNAreplicationforkprogression throughdamagedDNAbytheMec1/Rad53checkpoint. Nature 2001, 412 :553-557. 32.MerrillBJ,HolmC: Arequirementforrecombinationalrepairin SaccharomycescerevisiaeiscausedbyDNAreplicationdefectsofmec1 mutants. Genetics 1999, 153 :595-605. 33.GrushcowJM,HolzenTM,ParkKJ,WeinertT,LichtenM,BishopDK: SaccharomycescerevisiaecheckpointgenesMEC1,RAD17andRAD24 arerequiredfornormalmeioticrecombinationpartnerchoice. Genetics 1999, 153 :607-620. 34.KatoR,OgawaH: Anessentialgene,ESR1,isrequiredformitoticcell growth,DNArepairandmeioticrecombinationinSaccharomyces cerevisiae. NucleicAcidsRes 1994, 22 :3104-3112. 35.LydallD,NikolskyY,BishopDK,WeinertT: Ameioticrecombination checkpointcontrolledbymitoticcheckpointgenes. Nature 1996, 383 :840-843. 36.GreenwellPW,KronmalSL,PorterSE,GassenhuberJ,ObermaierB, PetesTD: TEL1,ageneinvolvedincontrollingtelomerelengthinS. cerevisiae,ishomologoustothehumanataxiatelangiectasiagene. Cell 1995, 82 :823-829. 37.RitchieKB,MalloryJC,PetesTD: InteractionsofTLC1(whichencodesthe RNAsubunitoftelomerase),TEL1,andMEC1inregulatingtelomere lengthintheyeastSaccharomycescerevisiae. MolCellBiol 1999, 19 :6065-6075. 38.SanchezY,DesanyBA,JonesWJ,LiuQ,WangB,ElledgeSJ: Regulationof RAD53bytheATM-likekinasesMEC1andTEL1inyeastcellcycle checkpointpathways. Science 1996, 271 :357-360.Doerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page12of13

PAGE 13

39.MeloJA,CohenJ,ToczyskiDP: Twocheckpointcomplexesare independentlyrecruitedtositesofDNAdamageinvivo. GenesDev 2001, 15 :2809-2821. 40.KondoT,WakayamaT,NaikiT,MatsumotoK,SugimotoK: Recruitmentof Mec1andDdc1checkpointproteinstodouble-strandbreaksthrough distinctmechanisms. Science 2001, 294 :867-870. 41.MakovetsS,BlackburnEH: DNAdamagesignallingpreventsdeleterious telomereadditionatDNAbreaks. NatCellBiol 2009, 11 :1383-1386. 42.ZhangW,DurocherD: Denovotelomereformationissuppressedbythe Mec1-dependentinhibitionofCdc13accumulationatDNAbreaks. Genes Dev 24 :502-515. 43.BashkirovVI,KingJS,BashkirovaEV,Schmuckli-MaurerJ,HeyerWD: DNA repairproteinRad55isaterminalsubstrateoftheDNAdamage checkpoints. MolCellBiol 2000, 20 :4393-4404. 44.FlottS,AlabertC,TohGW,TothR,SugawaraN,CampbellDG,HaberJE, PaseroP,RouseJ: PhosphorylationofSlx4byMec1andTel1regulates thesingle-strandannealingmodeofDNArepairinbuddingyeast. Mol CellBiol 2007, 27 :6433-6445. 45.EhmsenKT,HeyerWD: SaccharomycescerevisiaeMus81-Mms4isa catalytic,DNAstructure-selectiveendonuclease. NucleicAcidsRes 2008, 36 :2182-2195. 46.MordesDA,NamEA,CortezD: Dpb11activatestheMec1-Ddc2complex. ProcNatlAcadSciUSA 2008, 105 :18730-18734. 47.HerzbergK,BashkirovVI,RolfsmeierM,HaghnazariE,McDonaldWH, AndersonS,BashkirovaEV,YatesJR,HeyerWD: PhosphorylationofRad55 onserines2,8,and14isrequiredforefficienthomologous recombinationintherecoveryofstalledreplicationforks. MolCellBiol 2006, 26 :8396-8409. 48.ZhouZ,ElledgeSJ: DUN1encodesaproteinkinasethatcontrolsthe DNAdamageresponseinyeast. Cell 1993, 75 :1119-1127. 49.HuangM,ZhouZ,ElledgeSJ: TheDNAreplicationanddamage checkpointpathwaysinducetranscriptionbyinhibitionoftheCrt1 repressor. Cell 1998, 94 :595-605. 50.SanchezY,BachantJ,WangH,HuF,LiuD,TetzlaffM,ElledgeSJ: Control oftheDNAdamagecheckpointbychk1andrad53proteinkinases throughdistinctmechanisms. Science 1999, 286 :1166-1171. 51.GardnerR,PutnamCW,WeinertT: RAD53,DUN1andPDS1definetwo parallelG2/Mcheckpointpathwaysinbuddingyeast. EmboJ 1999, 18 :3173-3185. 52.ChanJE,KolodnerRD: Ageneticandstructuralstudyofgenome rearrangementsmediatedbyhighcopyrepeatTy1elements. PLoSGenet 7 :e1002089. 53.ChenQ,IjpmaA,GreiderCW: Twosurvivorpathwaysthatallowgrowth intheabsenceoftelomerasearegeneratedbydistincttelomere recombinationevents. MolCellBiol 2001, 21 :1819-1827. 54.TengSC,ChangJ,McCowanB,ZakianVA: Telomerase-independent lengtheningofyeasttelomeresoccursbyanabruptRad50p-dependent, Rif-inhibitedrecombinationalprocess. MolCell 2000, 6 :947-952. 55.TengSC,ZakianVA: Telomere-telomererecombinationisanefficient bypasspathwayfortelomeremaintenanceinSaccharomycescerevisiae. MolCellBiol 1999, 19 :8083-8093. 56.MalkovaA,IvanovEL,HaberJE: Double-strandbreakrepairinthe absenceofRAD51inyeast:apossibleroleforbreak-inducedDNA replication. ProcNatlAcadSciUSA 1996, 93 :7131-7136. 57.SignonL,MalkovaA,NaylorML,KleinH,HaberJE: Geneticrequirements forRAD51-andRAD54-independentbreak-inducedreplicationrepairof achromosomaldouble-strandbreak. MolCellBiol 2001, 21 :2048-2056. 58.SymingtonLS: RoleofRAD52epistasisgroupgenesinhomologous recombinationanddouble-strandbreakrepair. MicrobiolMolBiolRev 2002, 66 :630-670,tableofcontents. 59.SugawaraN,IraG,HaberJE: DNAlengthdependenceofthesingle-strand annealingpathwayandtheroleofSaccharomycescerevisiaeRAD59in double-strandbreakrepair. MolCellBiol 2000, 20 :5300-5309. 60.SchmidtKH,ViebranzE,DoerflerL,LesterC,RubensteinA: Formationof complexandunstablechromosomaltranslocationsinyeast. PLoSOne 2010, 5 :e12007. 61.PannunzioNR,MantheyGM,BailisAM: RAD59isrequiredforefficient repairofsimultaneousdouble-strandbreaksresultingintranslocations inSaccharomycescerevisiae. DNARepair(Amst) 2008, 7 :788-800. 62.OnodaF,SekiM,MiyajimaA,EnomotoT: Elevationofsisterchromatid exchangeinSaccharomycescerevisiaesgs1disruptantsandthe relevanceofthedisruptantsasasystemtoevaluatemutationsin Bloom ssyndromegene. MutatRes 2000, 459 :203-209. 63.OoiSL,ShoemakerDD,BoekeJD: DNAhelicasegeneinteractionnetwork definedusingsyntheticlethalityanalyzedbymicroarray. NatGenet 2003, 35 :277-286. 64.WattPM,HicksonID,BortsRH,LouisEJ: SGS1,ahomologueofthe Bloom sandWerner ssyndromegenes,isrequiredformaintenanceof genomestabilityinSaccharomycescerevisiae. Genetics 1996, 144 :935-945. 65.LouisEJ,HaberJE: ThestructureandevolutionofsubtelomericY repeats inSaccharomycescerevisiae. Genetics 1992, 131 :559-574. 66.LydeardJR,JainS,YamaguchiM,HaberJE: Break-inducedreplicationand telomerase-independenttelomeremaintenancerequirePol32. Nature 2007, 448 :820-823. 67.Fishman-LobellJ,HaberJE: RemovalofnonhomologousDNAendsin double-strandbreakrecombination:theroleoftheyeastultraviolet repairgeneRAD1. Science 1992, 258 :480-484. 68.IvanovEL,HaberJE: RAD1andRAD10,butnototherexcisionrepair genes,arerequiredfordouble-strandbreak-inducedrecombinationin Saccharomycescerevisiae. MolCellBiol 1995, 15 :2245-2251. 69.HwangJY,SmithS,MyungK: TheRad1-Rad10complexpromotesthe productionofgrosschromosomalrearrangementsfromspontaneous DNAdamageinSaccharomycescerevisiae. Genetics 2005, 169 :1927-1937. 70.BartschS,KangLE,SymingtonLS: RAD51isrequiredfortherepairof plasmiddouble-strandedDNAgapsfromeitherplasmidor chromosomaltemplates. MolCellBiol 2000, 20 :1194-1205. 71.MalagonF,AguileraA: Yeastspt6-140mutation,affectingchromatinand transcription,preferentiallyincreasesrecombinationinwhichRad51pmediatedstrandexchangeisdispensable. Genetics 2001, 158 :597-611. 72.TranPT,ErdenizN,SymingtonLS,LiskayRM: EXO1-Amulti-tasking eukaryoticnuclease. DNARepair(Amst) 2004, 3 :1549-1559. 73.SchmutteC,SadoffMM,ShimKS,AcharyaS,FishelR: Theinteractionof DNAmismatchrepairproteinswithhumanexonucleaseI. JBiolChem 2001, 276 :33011-33018. 74.TishkoffDX,BoergerAL,BertrandP,FilosiN,GaidaGM,KaneMF, KolodnerRD: IdentificationandcharacterizationofSaccharomyces cerevisiaeEXO1,ageneencodinganexonucleasethatinteractswith MSH2. ProcNatlAcadSciUSA 1997, 94 :7487-7492. 75.MorinI,NgoHP,GreenallA,ZubkoMK,MorriceN,LydallD: CheckpointdependentphosphorylationofExo1modulatestheDNAdamage response. EmboJ 2008, 27 :2400-2410. 76.ChenC,UmezuK,KolodnerRD: ChromosomalrearrangementsoccurinS. cerevisiaerfa1mutatormutantsduetomutageniclesionsprocessedby double-strand-breakrepair. MolCell 1998, 2 :9-22. 77.SchmidtKH,PennaneachV,PutnamCD,KolodnerRD: AnalysisofgrosschromosomalrearrangementsinSaccharomycescerevisiae. Methods Enzymol 2006, 409 :462-476. 78.GietzRD,WoodsRA: YeasttransformationbytheLiAc/SSCarrierDNA/ PEGmethod. MethodsMolBiol 2006, 313 :107-120. 79.LeaDE,CoulsonCA: Thedistributionofthenumberofmutantsin bacterialpopulations. JGenet 1949, 49 :264-285. 80.NairKR: Tableofconfidenceintervalsforthemedianinsamplesfrom anycontinuouspopulation. Sankhya 1940, 4 :551-558. 81.MirzaeiH,SyedS,KennedyJ,SchmidtKH: Sgs1truncations inducegenomerearrangementsbutsuppressdetrimentaleffectsofBLM overexpressioninSaccharomycescerevisiae. JMolBiol 2011, 405 :877-891. 82.SchmidtKH,ViebranzEB,HarrisLB,Mirzaei-SouderjaniH,SyedS,MedicusR: DefectsinDNAlesionbypassleadtospontaneouschromosomal rearrangementsandincreasedcelldeath. EukaryotCell 2010, 9 :315-324.doi:10.1186/2041-9414-2-8 Citethisarticleas: Doerfler etal .: Differentialgeneticinteractions betweenSgs1,DNA-damagecheckpointcomponentsandDNArepair factorsinthemaintenanceofchromosomestability. GenomeIntegrity 2011 2 :8.Doerfler etal GenomeIntegrity 2011, 2 :8 http://www.genomeintegrity.com/content/2/1/8 Page13of13