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Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenan...
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Title: Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenance of chromosome stability
Series Title: Genome Integrity
Physical Description: Archival
Language: English
Creator: Doerfler, Lillian
Publisher: BioMed Central
Publication Date: 2011
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Abstract: Background: Genome instability is associated with human cancers and chromosome breakage syndromes, including Bloom’s syndrome, caused by inactivation of BLM helicase. Numerous mutations that lead to genome instability are known, yet how they interact genetically is poorly understood. Results: We show that spontaneous translocations that arise by nonallelic homologous recombination in DNAdamage- checkpoint-defective yeast lacking the BLM-related Sgs1 helicase (sgs1Δ mec3Δ) are inhibited if cells lack Mec1/ATR kinase. Tel1/ATM, in contrast, acts as a suppressor independently of Mec3 and Sgs1. Translocations are also inhibited in cells lacking Dun1 kinase, but not in cells defective in a parallel checkpoint branch defined by Chk1 kinase. While we had previously shown that RAD51 deletion did not inhibit translocation formation, RAD59 deletion led to inhibition comparable to the rad52Δ mutation. A candidate screen of other DNA metabolic factors identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1Δ mutant, becoming even more important for chromosomal stability upon MEC3 deletion. We determined that the C-terminal third of Exo1, harboring mismatch repair protein binding sites and phosphorylation sites, is dispensable for Exo1’s roles in chromosomal rearrangement suppression, mutation avoidance and resistance to DNA-damaging agents. Conclusions: Our findings suggest that translocations between related genes can form by Rad59-dependent, Rad51-independent homologous recombination, which is independently suppressed by Sgs1, Tel1, Mec3 and Exo1 but promoted by Dun1 and the telomerase-inhibitor Mec1. We propose a model for the functional interaction between mitotic recombination and the DNA-damage checkpoint in the suppression of chromosomal rearrangements in sgs1Δ cells.
General Note: Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/ufir) at the time of funding. The Institutional Repository at the University of Florida (IR@UF) is the digital archive for the intellectual output of the University of Florida community, with research, news, outreach and educational materials
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Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: doi - 10.1186-2041-9414-2-8
System ID: AA00008938:00001

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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. 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