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G3: Genes|Genomes|Genetics Early Online, published on September 27, 2016 as doi:10.1534/g3.116.033910 PreventionofDNARereplicationThroughaMeioticRecombinationCheckpointResponse NicoleA.Najor,*,1LayneWeatherford,†andGeorgeS.Brush,†,‡,2 *DepartmentofPharmacology,WayneStateUniversitySchoolofMedicine,Detroit,MIUSA † DepartmentofOncology,WayneStateUniversitySchoolofMedicine,Detroit,MIUSA ‡ MolecularTherapeuticsProgram,BarbaraAnnKarmanosCancerInstitute,WayneState University,Detroit,MIUSA 1 Currentaddress:DepartmentofBiology,UniversityofDetroitMercy,Detroit,MIUSA 2 Correspondingauthor 1 © The Author(s) 2013. Published by the Genetics Society of America. Runningtitle: CheckingMeioticDNARereplication Keywords: Mec1,Rad53,recombination,DNAreplicationinitiation,proteinphosphorylation Correspondingauthor: GeorgeS.Brush,Ph.D. KarmanosCancerInstitute 3114PrentisCancerCenter 110E.WarrenAve. Detroit,MI48201 Tel:313-578-4300 Email:[email protected] 2 ABSTRACT InthebuddingyeastSaccharomycescerevisiae,unnaturalstabilizationofthecyclin-dependent kinaseinhibitorSic1duringmeiosiscantriggerextraroundsofDNAreplication.When programmedDNAdouble-strandbreaksaregeneratedbutnotrepairedduetoabsenceof DMC1,apathwayinvolvingthecheckpointgeneRAD17preventsthisDNArereplication. FurthergeneticanalysishasnowrevealedthatpreventionofDNArereplicationalsorequires MEC1,whichencodesaproteinkinasethatservesasacentralcheckpointregulatorinseveral pathwaysincludingthemeioticrecombinationcheckpointresponse.DownstreamofMEC1, MEK1isrequiredthroughitsfunctiontoinhibitrepairbetweensisterchromatids.Bycontrast, meioticrecombinationcheckpointeffectorsthatregulategeneexpressionandcyclindependentkinaseactivityarenotnecessary.PhosphorylationofhistoneH2A,whichiscatalyzed byMec1andtherelatedTel1proteinkinaseinresponsetoDNAdouble-strandbreaksandcan helpcoordinateactivationoftheRad53checkpointproteinkinaseinthemitoticcellcycle,is requiredforthefullcheckpointresponse.PhosphorylationsitesthataretargetedbyRad53in mitoticSphasecheckpointresponsesarealsoinvolved,basedonthebehaviorofcells containingmutationsintheDBF4andSLD3DNAreplicationgenes.However,RAD53doesnot appeartoberequired,nordoesRAD9,whichencodesamediatorofRad53,consistentwith theirlackoffunctionintherecombinationcheckpointpathwaythatpreventsmeiotic progression.Whilethisresponseissimilarinsomerespectstocheckpointmechanismsthat inhibitinitiationofDNAreplicationinthemitoticcellcycle,theevidencepointstoanew variationonDNAreplicationcontrol. 3 INTRODUCTION DNAreplicationduringmeiosisgeneratesthenecessarychromosomalcontentforthe subsequentformationofhaploidgametesthroughtwoconsecutiveroundsofchromosome segregation.Asduringthemitoticcellcycle,meioticDNAreplicationistightlyregulatedsothat initiationoccursatpreciselythecorrecttimeandonlyonceduringtheprocess(Strich2004);in theabsenceofappropriatecontrols,errorssuchasDNArereplicationcanoccurthatare typicallyharmfultothecell.Cyclin-dependentkinase(CDK)complexesarecentralregulatorsof eukaryoticDNAreplicationinitiation,bothinthemitoticcellcycle(Siddiquietal.2013)andin meiosis(Diricketal.1998;StuartandWittenberg1998;Benjaminetal.2003).Wehaveshown inSaccharomycescerevisiaethatexpressionofastabilizedformoftheB-typecyclin-CDK inhibitorSic1duringmeiosiscanleadtoextraroundsofDNAreplication(Sawarynskietal. 2009).Thisobservationisconsistentwiththewell-establishedroleofCDK,particularlyClb5Cdk1,inpreventingDNArereplicationduringthemitoticcellcyclethroughseveralmechanisms thatservetoinhibitreformationofthepre-replicativecomplex(Nguyenetal.2001;Ikuietal. 2007;Siddiquietal.2013). Asinmosteukaryoticorganisms,meioticDNAreplicationinS.cerevisiaeisfollowedby programmedrecombinationbetweenhomologouschromosomesduringprophaseofthefirst meioticdivision.Thephysicalinteractionofhomologsaffordedbyrecombinationisimportant foraccuratechromosomesegregationduringthisdivision,andallowsfortransferofgenetic informationbetweentheparentalchromosomes.MeioticrecombinationinitiatesfromaDNA double-strandbreak(DSB)generatedbySpo11,atopoisomerase-likeenzymewithDNA transesteraseactivitythatfunctionsincooperationwithseveralotherproteins(Keeneyetal. 1997;Malekietal.2007).ItisestimatedthatSpo11catalyzesformationof140-170DSBsper 4 meiosisinS.cerevisiae(Buhleretal.2007;Panetal.2011),withanumberofcontrolsinplace toensurethateachofthe16chromosomessustainsatleastoneevent(YoudsandBoulton 2011).EachDSBisinitiallyprocessedtogenerate3’-single-strandedDNAoverhangsthatcan invadethehomologousduplexchromosome(Caoetal.1990;Sunetal.1991).Intheabsenceof themeiosis-specificDNArecombinaseDmc1,strandinvasioncannotproceedandextensive DNAresectionresults,leadingtoactivationofameioticrecombinationcheckpointresponse thatpreventsexitfromthepachytenestageofprophaseI(Bishopetal.1992;Xuetal.1997). Asmightbeexpected,themeioticrecombinationcheckpointpathwayasdefinedby deletionoftheDMC1gene(dmc1Δ)sharesmanyproteinswithDNAdamagecheckpoint pathwaysthatoperateduringthemitoticcellcycle(Lydalletal.1996).Examplesincludethe apicalproteinkinaseMec1anditsassociatedproteinDdc2,whichareorthologsofhumanATM- andRad3-relatedproteinkinase(ATR)andATR-interactingprotein(ATRIP),respectively,and thePCNA-likeDdc1-Mec3-Rad17(“9-1-1”)complex,whichfacilitatesMec1functionandalso hasahumancounterpart(Weinertetal.1994;Lydalletal.1996;Paciottietal.2000;Hongand Roeder2002;ZouandElledge2003;Navadgi-PatilandBurgers2011;Refolioetal.2011).In addition,theDot1methyltransferaseisinvolvedinboth(San-SegundoandRoeder2000; Giannattasioetal.2005;Wysockietal.2005).Bycontrast,theproteinkinaseRad53,an orthologofhumanCHK2,anditsmediatorRad9,similarinsomerespectstohumanmediators suchasBRCA1and53BP1,functiondownstreamofMec1invariouscellcycleDNAdamage checkpoints(Allenetal.1994;Weinertetal.1994;Sunetal.1996;Gilbertetal.2001;Stracker etal.2009)butarenotinvolvedinthemeioticrecombinationcheckpoint(Bishopetal.1992; Lydalletal.1996;BailisandRoeder2000).WhileRad53andRad9havebeenimplicatedin certainmeioticcheckpoints,includingtheresponsetohydroxyurea(HU)(S-phase)(Blitzblau 5 andHochwagen2013)andtounprogrammedDNAdamage(WeberandByers1992;CartagenaLirolaetal.2008),theirabsenceintherecombinationcheckpointcanbeexplainedbythe existenceofmeiosis-specificproteinsthatoperatespecificallyinthecontextofrecombination intermediatestructures(HollingsworthandPonte1997;Xuetal.1997;BailisandRoeder2000). TheseincludeHop1,Red1,andMek1,eachofwhichisacomponentofthesisterchromatidderivedaxialelementsthatformduringmeiosisandarecriticalforpropermeiotic recombination.Hop1andRed1arestructuralinnature(Hollingsworthetal.1990;Smithand Roeder1997),whereasMek1isaproteinkinasewithsequencesimilaritytoRad53(Rockmill andRoeder1991;LeemandOgawa1992;BailisandRoeder2000).Allthreeproteinshelpto enforcetheproperbiasofinter-homologrecombinationduringunperturbedmeiosis,thereby promotingfaithfulchromosomesegregation(HollingsworthandByers1989;Rockmilland Roeder1991;SchwachaandKleckner1997;ThompsonandStahl1999;Kimetal.2010;Wuetal. 2010).InthecontextofthemeioticrecombinationcheckpointactivatedbydeletionofDMC1, Red1associateswiththe9-1-1complextohelpactivateMec1(EichingerandJentsch2010), leadingtoMec1-catalyzedHop1phosphorylationrequiredforMek1activation(Carballoetal. 2008).Mek1activityinturnpreventsexcessiverecombinationbetweensisterchromatidsand therebymaintainsthecheckpointsignal(Wanetal.2004;Niuetal.2005).Studiesusing mutantswithdefectsininter-sisterDSBrepair,orthoseinwhichtheDSBsthataregenerated cannotbeefficientlyrepaired,haveshownthatMek1alsoservestopreventmeiotic progression(Xuetal.1997;Cartagena-Lirolaetal.2008;Wuetal.2010). Ultimately,checkpoint-mediatedpreventionofpachyteneexitandprogressionthrough themeioticdivisionsisimplementedinpartthroughregulationoftheNDT80geneandits proteinproduct,whichisameiosis-specifictranscriptionfactorrequiredforproperexpression 6 ofmany“middle”sporulationgenes(ChuandHerskowitz1998;Hepworthetal.1998;Lindgren etal.2000;Tungetal.2000;PakandSegall2002;Shubassietal.2003).TheseincludeCDC5, whosepolo-likeproteinkinaseproductisrequiredforpachyteneexitandalsoup-regulates Ndt80activationinafeedbackloop(SourirajanandLichten2008;Acostaetal.2011),andCLB1, whichencodesaB-typecyclinthatisrequiredforprogressionthroughmeiosisI(Chuand Herskowitz1998;CarlileandAmon2008).Anothertargetofthemeioticrecombination checkpointistheSwe1proteinkinase,whichisactivatedtocatalyzeinhibitoryphosphorylation ofCdk1attyrosine19(LeuandRoeder1999).EarlyworkinmitoticcellsindicatedthatSwe1catalyzedCdk1phosphorylationregulatesthemorphogenesischeckpoint(LewandReed1995). However,itisnowknownthatSwe1isalsoacomponentofoneofthreeMec1-dependent mechanismsthatoperateintheSphasecheckpointtopreventcellcycleprogressioninto mitosis(Palouetal.2015). Inourpreviousstudies,wefoundthatdeletionofDMC1blocksDNArereplication inducedbySic1stabilization(Sawarynskietal.2009).Inthisreport,wedescribeourfurther geneticinvestigationintoconstituentsofthemeioticrecombinationcheckpointastheypertain toSic1-inducedDNArereplication.Wefoundthatcertainupstreamcomponents,including MEC1,wererequiredtopreventDNArereplication.However,wedidnotfindevidencethat particulardownstreameffectorsthatregulatemeioticprogressionwereinvolved.Wefurther examinedprocessesthatoperatetopreventDNAreplicationinthemitoticcellcycle,andfound overlapwithrespecttospecificphosphorylationevents,includingthosethatareimportantfor blockinglateDNAreplicationoriginfiringinSphasecheckpointresponses.Interestingly,these datasuggestapathwayinwhichtheeffectorsarephosphorylatedthroughaRad53independentmechanism. 7 MATERIALSANDMETHODS Strains.YeaststrainsusedinthisstudyarelistedinTable1.Constructionofthe HOP1pr-SIC1∆PHAmoduleanditsintegrationintothegenomeweredescribedpreviously (Sawarynskietal.2009).Inmostcases,deletionmutationsweregeneratedinhaploidsby homology-directedsite-specificreplacementwithselectablemarkers(Baudinetal.1993). ThesemarkerswerePCR-amplifiedfromeithergenomicDNAofadeletionsetmutant(Winzeler etal.1999)(GEDharmacon)orfromaplasmid(Brachmannetal.1998).Certainmutant progenitorstrainsintheW303backgroundweregenerouslyprovidedbyotherinvestigators: SKY2939(h2a-S129A)(Downsetal.2004)byStephenKron(UniversityofChicago),YFL234 (dot1Δ)(Giannattasioetal.2005)byMarcoMuzi-Falconi(UniversitàdegliStudidiMilano), U960(rad53Δsml1-1)(Zhaoetal.1998)byStephenElledge(HarvardUniversity),andY2359 andY2573(dbf4-4A,sld3-38A,andmcm5-bob1)(ZegermanandDiffley2010)byPhilip Zegerman(TheGurdonInstitute,UK)andJohnDiffley(TheFrancisCrickInstitute,UK).These mutationswerethenintroducedintoourcellsystemthroughcrossing.Strainconstruction generallyinvolvedintroductionofmutationsintoMATacellsandintoMATαcellswiththe HOP1pr-SIC1∆PHAmoduleeitherpresentorsubsequentlyadded,followedbymatingofthetwo celltypes.(NotethattheshorthanddesignationofSIC1∆PHAusedfordiploidsinthetextand figuresindicatesthepresenceofasinglecopyoftheHOP1pr-SIC1∆PHAelement,whileother mutantalleledesignationsindicatealterationofbothgenecopies.)Alldeletionmutations generatedforthisstudywereverifiedbyPCR,anddeletionofSWE1wasfurtherconfirmedby westernblottingusingantibodykindlyprovidedbyDougKellogg(UniversityofCalifornia,Santa Cruz)(SreenivasanandKellogg1999).DNAsequencingwasusedtovalidatethepresenceof certainpointmutationsinourstrains.EpitopetaggingofSic1(SIC113MYC)wasperformedas 8 described(Longtineetal.1998)inMATaandMATαcells,whichwerethenmatedtogenerate thediploid.Anadditionalmanipulationincluded5-fluoro-oroticacid-mediatedcounterselection(Boekeetal.1984)toisolatearad53Δsml1-1diploidfromastraincontainingHOP1prSIC1ΔPHA. Cellculture.Allyeastincubationswereconductedat30°C.Meiosiswasinducedby starvationbasedonanestablishedprocedureforsynchronoussporulation(Padmoreetal. 1991).Inthismethod,yeastcellswerefirstgrownonsolid(2%(w/v)agar)YPGmedium(1% (w/v)yeastextract,2%(w/v)peptone,3%(v/v)glycerol)(oralternativelyonsolidYPDmedium (1%(w/v)yeastextract,2%(w/v)peptone,2%(w/v)dextrose)for3-4days,andsinglecolonies wereusedtoinoculateYPDliquidcultures.TheovernightYPDcultureswerethenusedto inoculateYPA(1%(w/v)yeastextract,2%(w/v)peptone,2%(w/v)potassiumacetate)atan OD600of~0.2.Cellswereincubatedovernight,typicallyfor16hours,andthenresuspendedat equivalentcelldensities(basedonOD600values)forstrainswithinanexperimentinsporulation mediumconsistingof0.3%(w/v)potassiumacetateand0.02%(w/v)raffinosesupplemented withleucine,arginine,andhistidineeachat250μM,tryptophanat100μM,anduracilat50μM. Cellswerereturnedtoincubationattime0andaliquotswereharvestedatindicatedtime pointsforflowcytometryandproteinanalyses(seebelow).Formostexperiments,control strainsSIC1∆PHAandSIC1∆PHAdmc1∆wereincluded.Eachexperimentalstrainwasanalyzedat leasttwiceinindependentexperiments,andinmanycasesmorethantwice(seeFigureS1). Toconductasynchronizedmitoticcellcycletimecourse,MATacellsgrowntosaturation inYPDwerebroughttoanOD600of~0.2andincubatedfor2hours.Theyeastmating pheromoneα-factor(ZymoResearch)wasthenaddedtoafinalconcentrationof2.5µMand 9 cellswereincubatedforanadditional2hourstoachieveG1arrest.Thecellswerethenwashed withsterilewatertoremoveα-factor,resuspendedinfreshYPD,andfurtherincubated. Aliquotsweretakenat15-minuteintervalsforflowcytometryandwesternblottinganalyses (seebelow).ForexaminingtheresponsetoinhibitionofDNAreplication,cellswerearrested withα-factorasdescribedaboveandthenreleasedinto0.8XYPDcontaining0.2MHU(MP Biomedicals). DNAcontent.Cellswereharvestedbycentrifugation,resuspendedin70%ethanoland storedat4°C.Aliquotsofthefixedcellswerewashedoncewith50mMTris–HCl,pH7.5, resuspendedin1mlofthesamebuffer,andthentreatedwith250μgRNaseAfor1hourat 37°Cfollowedby250μgproteinaseKfor1hourat37°C.Digestedsampleswereincubatedwith 10XSYBRGreenI(MolecularProbes)at4°Covernight,sonicatedbrieflyandanalyzedwitha FACSCantoIIflowcytometer(BDBiosciences)(or,inoneexperiment,aBDLSRIIflow cytometer)(Microscopy,Imaging,andCytometryResourcesCoreatWayneStateUniversity SchoolofMedicine).DNAcontenthistogramsweregeneratedusingWinMDIfreeware.DNA rereplicationwasquantifiedbyusingthegatingfunctioninWinMDItodeterminethenumber ofevents(outof20,000)thatwererecordedwith>4CDNAcontent.Gatingwasestablished basedonthe4CDNApeakandwasheldconstantwithineachindividualexperiment. Protein.Cellswereharvestedbycentrifugationandstoredat-70oC.Formost experiments,denaturedwhole-cellextractswerepreparedbasedonanalkalineextraction method(Kushnirov2000).InthecaseofRad53analysis,atricholoroaceticacidbeadbeating methodwasused,asdescribed(Foianietal.1994).ResultingsamplesweresubjectedtoSDS 10 polyacrylamideelectrophoresis.Forwesternblotting,theseparatedproteinsweretransferred tonitrocellulose(GEHealthcare).Primaryantibodiesincludedratanti-α-tubulin(Serotec), mouseanti-hemagglutinin(Covance),rabbitanti-yeastγ-H2A(generouslyprovidedby ChristopheRedonandWilliamBonner,NationalCancerInstitute)(Nakamuraetal.2006), mouseanti-myc(SantaCruz),andrabbitanti-Rad53(Abcam).Signalsweregeneratedwith IRDye800-conjugatedgoatanti-rat(Rockland),AlexaFluor680goatanti-rabbit(Invitrogen),or AlexaFluor680goatanti-mouse(Invitrogen)secondaryantibodies.Reactivebandswere visualizedwithanOdysseyinfraredimagingsystem(Li-Cor).ForanalysisofRad53activity, separatedproteinsweretransferredtoPVDF(Millipore)andRad53autophosphorylationinsitu wasanalyzedasdescribed(Foianietal.1994).Inthefigures,linebordersindicatecropping,and verticallycontiguouspanelsindicatedataoriginatingfromthesameblotormembrane. 11 RESULTS Wehavedevelopedasysteminwhichcellsundergoingmeiosisexperienceatleastone extraroundofDNAreplication(Sawarynskietal.2009).ThisphenotypeoccursuponderegulationofCDKactivitythroughearlymeiosis-specificexpression(viatheHOP1promoter)of SIC1∆PHA,encodingaSic1HAvariantlackingcriticalCDKphosphorylationsitesthatarenecessary foritsdestruction(Vermaetal.1997).DNArereplicationdoesnotoccurinthissystemwhen theSic1HAphosphorylationsitesarenotaltered,asthisversionoftheproteinissubjecttoposttranslationalmodificationanddegradation.Interestingly,deletionofDMC1(dmc1Δ)activatesa responsethatpreventstheDNArereplicationphenotypenormallyobservedinourspecially engineeredcells.Wehaveexaminedthispathwaygenetically,asdescribedbelow.Theextent ofDNArereplicationinthevariousstrainsthatwedescribeinthisstudywasquantifiedfrom flowcytometrydata,andispresentedasacompilationinFigureS1. FigureS1.QuantificationofDNArereplication.Foreachexperiment,thesampleafter overnightincubation(24hrinmostcases)wasanalyzedfor>4CDNAcontentusingthe gatingfunctioninWinMDIsoftware.A,Asshowninthisexample,thegatingwasheld constantforeachsampleinanindividualexperiment.B,CompilationofDNA rereplicationresults.Thenumberofcellscontaining>4CDNAcontentoutof20,000 totalcellscountedisshownonthey-axis.Datapointsfromstrainsthatwere determinedtoexhibitDNArereplicationareshownasfilledcircles,whilethosefrom strainsdeterminednottoexhibitDNArereplicationareshownasopencircles.Innearly allexperiments,DNAcontentanalysiswasaccompaniedbyproteinanalysis.Itisnoted thatthreeofthe16SIC1∆PHAdmc1∆rad9∆repeatsdidshow>4CDNAcontent;inone 12 case,theSIC1∆PHAdmc1∆didaswell,andintheothertwocases,aberrant>4C“tailing” profileswereobserved.Anotherstrainofparticularinterest,SIC1∆PHAdmc1∆dbf4-4A sld3-38A,exhibitedvariability.However,inthetwoexperimentsofsixinwhichthis strainexhibitedonlybackgroundlevelsof>4CDNAcontent,progressionthrough normalDNAreplicationwasslow.ItispossiblethatlessrobustDNArereplicationis moresensitivetosubtleexperimentalvariations. MEC1.Previously,weshowedthatRAD17,whichencodesa9-1-1member,isrequired tosuppressdmc1Δ-dependentinhibitionofDNArereplication,suggestingthatabranchofthe meioticrecombinationcheckpointcouldaffectDNAreplication(Sawarynskietal.2009).Given thatRad17isintimatelyassociatedwithMec1,wesuspectedthatthiscentralproteinkinase wasalsoinvolved.Totestthishypothesis,wegeneratedamec1nullmutantinourstrain background.BecauseMEC1isessentialforviability(KatoandOgawa1994),itwasnecessaryto deleteSML1aswelltosuppressthelethalityresultingfromMEC1loss(Zhaoetal.1998).As showninFigure1,DNArereplicationwasobservedinSIC1∆PHAdmc1∆mec1∆sml1∆cells.This phenotypewasnotduetosml1∆,asSIC1∆PHAdmc1∆sml1∆cellsdidnotexhibitDNA rereplication(seeFigureS1).Therefore,MEC1wasinvolvedinthepreventionofDNA rereplicationinSIC1∆PHAdmc1∆cells.WeobservedlessrobustDNArereplicationinSIC1∆PHA dmc1∆mec1∆sml1∆cellsthanSIC1∆PHAcells;aportionofthiseffectmayhavebeenduetothe absenceofMEC1andSML1asrevealedbyexaminationofSIC1∆PHAmec1∆sml1∆cells(Figures 1andS1). 13 Figure1.MEC1isrequiredfordmc1Δ-dependentinhibitionofSIC1∆PHA-inducedDNA rereplication.Strainsweretreatedtoenterthemeioticprograminasynchronous fashion.Attheindicatedtimepoints,sampleswereanalyzedforSic1ΔPHA(HA)andthe tubulincontrol(tub)bywesternblotting(upperpanels)andforDNAcontentbyflow cytometry(lowerhistograms).Forthisfigureandothersthatfollow,the4Cdesignation indicatesthepopulationofcellsthathaveundergoneoneroundofDNAreplication; cellswithover-replicatedDNAappeartotherightofthisposition,andpeakswith approximateDNAcontents>4Careindicated.Conclusionsforthisexperimentare providedasschematicstoprovideanexampleofpathwayanalysis. MEK1andassociatedgenes.Wefurtherexaminedgenesthatoperatedownstreamof MEC1intheestablishedresponsethatpreventsbothinter-sisterrepairandmeioticprogression, includingthosethatencodetheaxialproteinsMek1,Red1,andHop1.Asinthecaseofmec1∆, deletionofanyoneofthesegenesrestoredDNArereplicationinSIC1∆PHAdmc1Δcells(Figure 2A-C),indicatingthattheywererequiredforpreventionofDNArereplicationinresponsetothe accumulationofunrepairedDSBs.Inthesecases,DNArereplicationwasrobust,asexhibitedby thegenerationofcellswithhighDNAcontentinsomecasesreaching~16C(Figures2andS1). Figure2.Factorsthatenforceinter-homologbiasandpreventmeioticprogressionare requiredfordmc1Δ-dependentinhibitionofSIC1∆PHA-inducedDNArereplication.Cells weretreatedtoentermeiosisandanalyzedforproteinlevelsbywesternblottingand DNAcontentbyflowcytometry.AandB,theeffectofred1∆andhop1∆,respectively;C, 14 theeffectofmek1∆,aswellasrad54∆.WesternblottinganalysisincludedSic1ΔPHA(HA), tubulin(tub)andphosphorylatedH2A(γ-H2A). Ithasbeenshownthatdmc1∆mek1∆cellsthatarealsorad54∆andthereforeincapable ofcompletinginter-sisterrepair(Arbeletal.1999)progressthroughmeiosis,albeitwithslower kineticsthanwild-typeordmc1Δmek1Δcells(Cartagena-Lirolaetal.2008;Chuangetal.2012); thisphenotypeillustratesthecheckpointfunctionofMEK1.TodeterminewhethertheMEK1 functiontosuppressinter-sisterrepairorthattopreventmeioticprogressionwasatplayinour specializedcase,weexaminedSIC1∆PHAdmc1∆mek1∆rad54∆cells.Wefoundthatthe recoveryofDNArereplicationobservedinSIC1∆PHAdmc1Δmek1Δcellswasnotobservedwith theadditionofrad54∆(Figure2C).Ineachstrain,weobservedanincreaseinphosphorylated histoneH2A(γ-H2A),whichisgeneratedthroughMec1(andrelatedTel1)catalysisinresponse toDSBformationandleadstoextensiveregionsofchromatincontainingγ-H2Aoneitherside oftheDSB(Shroffetal.2004).ThesedatasuggestedapersistenceofDSBsinourcells throughoutthetimecourseregardlessofDMC1orRAD54status.Wefurtherdemonstrated thatRAD54itselfwasnotrequiredfortheDNArereplicationphenotype(FigureS2).Thesedata suggestthatMEK1inhibitedDNArereplicationbypreventinginter-sisterrepairandmaintaining theDSB-inducedsignalratherthanbyinfluencingDNAreplicationitself. FigureS2.SIC1∆PHA-mediatedDNArereplicationoccursinrad54∆orh2a-S129A(γ-H2Anegative)cells.Theindicatedstrainswereallowedtoentermeiosisandthenanalyzed forSic1ΔPHA(HA),tubulin(tub),andγ-H2AbywesternblottingandDNAcontentbyflow cytometry. 15 StudieshaveindicatedthattheAAA+-typeATPasePch2suppressesinter-sisterrepairto someextentandhelpstopreventmeioticprogressionwhenunrepairedDSBsaccumulate(SanSegundoandRoeder1999;HoandBurgess2011;Zandersetal.2011;Chenetal.2014).We foundthatdeletionofPCH2relievedthedmc1∆-dependentinhibitionofDNArereplicationin ourSIC1∆PHAsystem(FigureS3),indicatingthatPCH2aidedinpreventingDNArereplication.In thiscase,asincertainothermutantsanalyzed(seebelow),fewcellswithDNAcontent>~8C wereobserved.ExtensiveDNArereplicationwasdetectedwithSIC1∆PHApch2∆cells(FiguresS3 andS1),indicatingthatPCH2itselfwasnotrequiredfortheDNArereplicationphenotypein SIC1∆PHAcells. FigureS3.PCH2promotesdmc1Δ-dependentinhibitionofSIC1∆PHA-inducedDNA rereplication.Theindicatedstrainswereallowedtoentermeiosisandthenanalyzedfor Sic1ΔPHA(HA)andtubulin(tub)bywesternblottingandDNAcontentbyflowcytometry. CDK.WeconsideredthepossibilitythatthecheckpointresponsemightpreventCDKactivation toinhibitDNArereplication.Onemechanismbywhichthemeioticrecombinationcheckpoint preventsmeioticprogressionisthroughSum1,akeytranscriptionfactorthatrepresses expressionofmiddlesporulationgenesnormallyinducedbythetranscriptionfactorNdt80 (Lindgrenetal.2000;PakandSegall2002;Winter2012).IncludedamongtheseNdt80-induced genesarethosethatencodetheB-typecyclinsClb1,-3,-4,-5,and-6(ChuandHerskowitz 1998).WefoundthatDNArereplicationoccurredinSIC1∆PHAsum1Δcells,althoughwith 16 reducedefficiencyforcellswithDNAcontent>~8C,butnotinSIC1∆PHAdmc1Δ sum1Δcells (Figures3AandS1),indicatingthatSUM1wasnotinvolvedinthischeckpoint. Figure3.RegulatorsofCdk1activationarenotrequiredfordmc1Δ-dependentinhibition ofDNArereplication.CellsweretreatedtoentermeiosisandexaminedforSic1ΔPHA (HA)andtubulin(tub)bywesternblottingandDNAcontentbyflowcytometry.A,the effectofsum1∆;BandC,theeffectofswe1∆.NotethattheSIC1ΔPHAdmc1ΔcontrolinA isidenticaltothatshowninFigure1. WealsoexaminedSWE1,whoseproductbecomesactivatedinthemeiotic recombinationcheckpointresponsetocatalyzeinhibitoryphosphorylationofCdk1attyrosine 19(LeuandRoeder1999).WefoundthatdeletionofSWE1,likethatofSUM1,didnotprevent DNArereplicationinSIC1∆PHAcells,nordiditreversethedmc1Δ-dependentinhibitionofDNA rereplication(Figure3BandC),suggestingthatSWE1wasnotrequiredforpreventionofDNA rereplication.ItisnotedthatbecauseNdt80levelsandactivityaredownregulatedbythe meioticrecombinationcheckpointresponse,therebyloweringB-typecyclinavailabilityandCDK activity,wemightnotexpecttoseemuchofaneffectbysimplydeletingSWE1inourcells. However,dmc1∆swe1∆cellsdoprogressintoMI,althoughwithdelayedkineticsrelativeto wildtypecells(LeuandRoeder1999).Duringthecourseofthesestudies,wealsoexamineda swe1ΔmutantwithouttheSIC1∆PHAallele.Thisexperimentwaspromptedbythereportthat swe1ΔcellsrereplicatetheirDNAduringmeiosis,aphenotypethatisdifferentfromoursinthat multisporeasciareformed(Riceetal.2005).Wedidnotobservethesephenotypesinour 17 swe1Δcells,perhapsduetodifferencesinstraintypesorcultureconditions(Figure3Banddata notshown). ToinvestigateanotherCDKregulator,Sic1,wegeneratedstrainsinwhichSic1was taggedwithMYCepitoperepeats.Inthisway,wecoulddistinguishendogenousSic1fromthe inducedversionlackingCDK-targetedphosphorylationsites,whichistaggedwiththeHA epitope.WefirstexaminedahaploidstrainduringthecellcycletoensurethatourSic113MYC proteinbehavedproperly.Asexpected,wefoundthatSic1disappearedalmostcompletelyas cellsprogressedfromG1arrest,andthenreappearedafterSphasewascompleted(FigureS4). Wenextexamineddiploidstrainsinmeiosis(Figure4).Synchronyisdifficulttoachievein meiosis,andwedidnotobservethesharpreductionandreappearanceofSic1inourwild-type cells.However,wedidseeadeclineinSic1ascellsprogressedthroughthetimecourse,which mayhavereflectedarealdecreaseincellularSic1steadystatelevels.Asshown,weobserved nearlyidenticalSic113MYCprofilesinSIC1∆PHAandSIC1∆PHAdmc1Δcells.Weconcludethata decreaseinCDKactivitythroughstabilizationofendogenousSic1wasnotlikelytobe responsiblefordmc1Δ-dependentinhibitionofDNArereplication. FigureS4.MYC-taggedSic1disappearsduringSphaseinmitoticcells.Haploidwild-type cells(SIC1)orthosecontainingSic1replacedwithanepitope-taggedversioncontaining 13MYCepitoperepeatsattheC-terminus(SIC113MYC)werearrestedinG1withα-factor andthenreleasedintothecellcycle.Attheindicatedtimepoints,sampleswere analyzedforSic113MYC(MYC)andtubulin(tub)bywesternblottingandforDNAcontent byflowcytometry. 18 Figure4.TheCDKinhibitorSic1doesnotaccumulateinSIC1∆PHAdmc1Δcells.The indicatedcellscontainingSic113MYCweretreatedtoentermeiosisandthenanalyzedat severaltimepointsforSic1ΔPHA(HA),Sic113MYC(MYC)andtubulin(tub)bywestern blottingandDNAcontentbyflowcytometry. γ-H2AandDOT1.Mec1-andTel1-catalyzedH2Aphosphorylationatserine129,which generatesγ-H2A,isthoughttobeanimportantprotectivemechanismthatpromotesDSB repairtopreventgenomicalterations(Downsetal.2000;Redonetal.2003)andfunctionsin theG1DNAdamagecheckpointduringthemitoticcellcycle(Javaherietal.2006;Hammetetal. 2007).BecauseMEC1wasrequiredinthemeioticrecombinationcheckpointresponsethat preventsDNArereplication,wesuspectedthatoneofitsmajortargetswouldbeinvolvedas well.Wefoundthatγ-H2AwasgeneratedinH2A(HTA1andHTA2)cellsregardlessofDMC1 status(seeFigure2C).WegeneratedaSIC1ΔPHAstrainwithHTA1andHTA2bothmutated(h2aS129A)sothatthetwoH2Asubunitscouldnotbephosphorylatedatserine129,andconfirmed throughwesternblottingthatthesecellsweredevoidofγ-H2A(FigureS5andseeFigureS2). Importantly,absenceofγ-H2AledtoDNArereplicationinSIC1∆PHAdmc1Δcells;whilenotas extensiveasinSIC1∆PHAcellswithregardtototalnumberofcellsexhibiting>4CDNAcontent andthoseexhibiting>~8C,theDNArereplicationwasclearandconsistentlyobserved(Figures5 andS1).SIC1∆PHAh2a-S129Acellsre-replicatedtheirDNAwithclearevidenceofcells containing>~8CDNAcontent(FigureS2). 19 FigureS5.SIC1ΔPHAdmc1Δh2a-S129Acellsaredevoidofγ-H2A.Theindicatedstrains weretreatedtoentermeiosisandthenanalyzedforγ-H2Aandtubulin(tub)bywestern blotting. Figure5.γ-H2AandDOT1areinvolvedindmc1Δ-dependentinhibitionofSIC1∆PHAinducedDNArereplication.Theindicatedstrainsweretreatedtoentermeiosisandthen analyzedforSic1ΔPHA(HA)andtubulin(tub)bywesternblottingandDNAcontentby flowcytometry. WealsoexaminedthephenotyperesultingfromdeletionofDOT1,whichencodesa histonemethyltransferaserequiredforthemeioticrecombinationcheckpointresponse(SanSegundoandRoeder2000).Dot1catalyzesmethylationofhistoneH3atlysineK79(H3meK79) (Lacosteetal.2002;Ngetal.2002;vanLeeuwenetal.2002)and,likeγ-H2A,isimportantfor theG1DNAdamagecheckpointinthemitoticcellcycle(Giannattasioetal.2005;Wysockietal. 2005).Similartothecasewithh2a-S129A,weobservedamodestdegreeofDNArereplication inSIC1∆PHAdmc1Δdot1∆cells,andcombinationofthedot1∆andh2a-S129Amutationsdidnot enhancethiseffect(Figures5andS1).Thesedatasuggestthatγ-H2AandDot1operatedinthe samepathwayinpreventingDNArereplicationinSIC1∆PHAdmc1Δcells. RAD53.WhileRad53isnotinvolvedinthemeioticrecombinationcheckpointperse,it canbeactivatedbygenotoxicstressduringmeiosis(WeberandByers1992;Cartagena-Lirolaet al.2008;BlitzblauandHochwagen2013).Therefore,weelectedtodeterminewhetherornot RAD53wasinvolvedinthecheckpointthatpreventsDNArereplicationinoursystem.(Asinthe 20 caseofmec1Δ,rad53ΔcellsareviablewhenSML1isalsodefective(Zhaoetal.1998).)Wewere surprisedtofindthatDNArereplicationdidnotoccurinSIC1∆PHArad53∆sml1-1cells, regardlessofDMC1status(FigureS6),evenafter48hrs(datanotshown).Asanalternative genetictest,weturnedtoRAD9,whichencodesaproteinthatmediatesRad53activationin manycircumstances(Sunetal.1998;Vialardetal.1998;Gilbertetal.2001).Bycontrastto rad53Δ,rad9∆didnotpreventDNArereplicationinSIC1∆PHAcells(FigureS6).Importantly, RAD9wasnotrequiredforsuppressingDNArereplicationinSIC1∆PHAdmc1Δcells(Figure6), indicatingthatRAD9wasnotinvolvedinthischeckpointresponse. FigureS6.DeletionofRAD53,butnotRAD9,abolishesSIC1∆PHA-inducedDNA rereplication.Theindicatedstrainsweretreatedtoentermeiosisandthenanalyzedfor Sic1ΔPHA(HA)andtubulin(tub)bywesternblottingandDNAcontentbyflowcytometry. A,theeffectofrad53∆;B,theeffectofrad9∆. Figure6.Rad53isnotactivatedbydeletionofDMC1.A,theindicatedstrainswere treatedtoentermeiosisandthenanalyzedfor:A,Sic1ΔPHA(HA)andtubulin(tub)by westernblottingandDNAcontentbyflowcytometry.B,Samplesfromthesametime courseshowninAwereusedtoassessRad53activationthroughwesternblotting (Rad53andtubulin(tub)toppanel)andRad53autophosphorylationinsitu(32P-Rad53, middlepanel).Totalproteinloadingfortheautophosphorylationassaywasdetermined byPonceauSstaining(bottompanel).ControlsamplesincludedvegetativeSIC1ΔPHA dmc1Δ(RAD53)andrad53Δsml1-1(rad53Δ)diploidsexposedtoHUfortheindicated times. 21 BecauseourresultsprecludedanalysisofRad53functionthroughgenedeletion,we exploredthepossibilityofaRad53orRad53-likefunctionthroughdifferentmeans.Inresponse toreplicationforkstallingorDNAdamageduringSphaseofthemitoticcellcycle,Rad53is activatedtocatalyzephosphorylationofDbf4andSld3,therebypreventingfiringoflateorigins (Lopez-Mosquedaetal.2010;ZegermanandDiffley2010).Weexaminedtwodifferenttypesof mutantstodeterminewhetherthisprocesscouldbeinvolvedinoursystem.Thefirstinvolved cellscontainingmutantallelesofbothDBF4andSLD3,encodingproteinswithalterationsin importantRad53-targetedphosphorylationsites.SIC1∆PHAdmc1∆cellscontainingthedbf4-4A andsld3-38AallelesexhibitedDNArereplication,indicatingthatphosphorylationofDbf4or Sld3,orbothproteins,wasrequiredforfullpreventionofDNArereplication(Figures7andS1). Wenoticedthat,asinthecaseofSIC1∆PHAdmc1∆h2A-S129Acells,thesecellsappearedto rereplicatetheirDNAlessthanSIC1∆PHAcellsinthattheydidnotexhibit>~8CDNAcontent.In SIC1∆PHAdbf4-4Asld3-38Acells,wedidobservecellswith>~8CDNAcontent,indicatingthat thephosphorylationsitemutationsthemselveswerenotresponsibleforlimitingDNA rereplication(FigureS7).Thesecondtypeofmutantcellscontainedthesld3-38Aalleleand mcm5-bob1,amutantallelethatbypassestheessentialfunctionofDBF4(Hardyetal.1997). DNArereplicationwasnotobservedinSIC1∆PHAdmc1∆mcm5-bob1sld3-38Acells(Figure7). Weconfirmedthatthemcm5-bob1alleledidnotpreventDNArereplicationinoursystem (FigureS7).WhileitmightbeexpectedthatcellswithalteredDbf4phosphorylationsiteswould behavesimilarlytocellswithmcm5-bob1,asinthemitoticcellcyclestudies,certain experimentalfactorsmayaccountforthisdiscrepancy(seeDiscussion).Thesedatasuggestthat Dbf4phosphorylationwassufficienttopreventDNAre-replication,atleastinthecontextofthe 22 mcm5-bob1allele.ItisnotedthateitherDbf4orSld3phosphorylationalonecancontributeto preventionoflateoriginfiringinmitoticSphase(Lopez-Mosquedaetal.2010;Zegermanand Diffley2010).WeconcludethatsitesnormallyphosphorylatedbyRad53inthemitoticcellcycle alsofunctionedtopreventDNAreplicationduringmeiosisunderourconditionsthatpromote DNArereplication. FigureS7.MutationsinDBF4andSLD3orMCM5andSLD3donotpreventSIC1∆PHAinducedDNArereplication.Theindicatedstrainsweretreatedtoentermeiosisandthen analyzedforSic1ΔPHA(HA)andtubulin(tub)bywesternblottingandDNAcontentby flowcytometry.SIC1∆PHAiscomparedwithA,SIC1∆PHAdbf4-4Asld3-38A,andB, SIC1∆PHAmcm5-bob1sld3-38A. TofurtherinvestigateapossibleRad53function,weexaminedRad53enzymaticactivity. WhileRad53isoformswithreducedelectrophoreticmobilityareindicativeofits phosphorylationandactivation,amoresensitivemethodistoanalyzeRad53 autophosphorylationinsitu(Pelliciolietal.1999).AscanbeseeninFigure6,aslightRad53 activationwasobservedatthelatetimepointintheSIC1ΔPHA,SIC1ΔPHAdmc1Δ,andSIC1ΔPHA dmc1Δrad9Δstrains.ThisactivationwasnegligiblerelativetoHU-inducedRad53activationin mitoticcells.WhileitappearedthattheRad53activationwasabithigherintheSIC1ΔPdmc1Δ cells,itisunlikelythatthisdegreeofactivationcouldhavebeenresponsibleforthecheckpoint giventhatheightenedactivationwasnotobservedinthecheckpoint-proficientSIC1ΔPHAdmc1Δ 23 rad9Δcells.ItispossiblethatpersistenceofDSBsandaccumulationofDNAdamageovertime inSIC1ΔPHAdmc1ΔcellsledtolowlevelRad9-dependentRad53phosphorylation. Figure7.SitesofphosphorylationtargetedbyRad53inthemitoticcellcycleinfluence dmc1Δ-dependentinhibitionofSIC1∆PHA-inducedDNArereplication.Theindicated strainsweretreatedtoentermeiosisandthenanalyzedforSic1ΔPHA(HA)andtubulin (tub)bywesternblottingandDNAcontentbyflowcytometry. 24 DISCUSSION WhenS.cerevisiaecellsdeletedforDMC1areinducedtoenterthemeiotic program,aresponseisactivatedthatinhibitsrecombinationbetweensisterchromatids andpreventsprogressionintothemeioticdivisions(Bishopetal.1992;Xuetal.1997; Wanetal.2004;Niuetal.2005).Wehaveobservedthatcellsundergoingmeiosisbut engineeredtorereplicatetheirDNAthroughexpressionofSIC1∆PHAalsorespondto deletionofDMC1bypreventingextraDNAreplication(Sawarynskietal.2009).Our previousstudiesindicatedthatthisresponserequiresRAD17,suggestingacheckpoint mechanism.Ourfurthergeneticanalysisshownherehasconfirmedthatacheckpoint responseisinvolved. WeobserveddifferencesinthedegreeofDNArereplicationrecoveryinSIC1∆PHA dmc1Δcellsdependingonwhichadditionalgenewasdeletedormutated.While experimentalvariabilitymakesitdifficulttobeconclusiveaboutthesedifferences,we observedtrendsinparticularcircumstances.Forexample,removalofgenesencoding certainproteins,suchasMek1,thatservetopreventbothinter-sisterrepairandmeiotic progressionledtoextensiveDNArereplicationwithcellscontaining>~8CDNAcontent. OurexperimentsfurthersuggestthattheMEK1functiontopreventinter-sisterrepair, andtotherebyretainthecheckpointsignaloriginatingfromunrepairedDSBs,is operativeinpreventingDNArereplicationinourcells.Bycontrasttothesegene deletions,mutationofgenestoabrogateH2AorDbf4andSld3phosphorylationledto lessDNArereplication,withfewcellsdetectedcontaining>~8CDNAcontent.This differencemightindicatethatthedegreeofDNArereplicationislimitedbythepresence ofunrepairedandresectedDSBs,althoughwedidobserveconsiderableγ-H2Astaining 25 incellsregardlessofcheckpointstatus.Inaddition,morethanasinglecheckpoint mechanismmaybeinvolvedinpreventingDNArereplication,withonlyonebeing absentincertaincasessuchasSIC1∆PHAdmc1Δdbf4-4Asld3-38Acells.Inthissense, perhapsthecheckpointpathwaythatwehaveuncoveredaffectsonlyasubsetoforigins. Regardless,thedataprovideclearevidencethatthemeioticrecombinationcheckpoint cantargettheDNAreplicationmachinery. ThemeioticrecombinationcheckpointresponsethatpreventsDNArereplication sharesseveralcomponentswithcellcycleDNAdamageresponsecheckpoint mechanisms.GenesencodingMec1andRad17,andpresumablytheentire9-1-1 complexthatincludesRad17andfacilitatesMec1function,wererequiredinoursystem. ItisinterestingtonotethatDNArereplicationitselfinthemitoticcellcycleinducesa checkpointresponsedependentonMEC1andRAD17thatrestrictstheextentofDNA rereplication(Archambaultetal.2005).Inourcase,weobservedslightlylessDNA rereplicationupondeletionofMEC1andSML1inSIC1∆PHAdmc1Δcellswhencompared withSIC1∆PHAcellsdeletedforgenessuchasMEK1,particularlywithregardto>~8C DNAcontentcells.(ThepossibilityisnotedthatdeletionofMEC1andSML1mighthave hadaminoreffectonDNArereplicationinSIC1∆PHAcellsaswell).Ithasbeenreported thatdmc1Δmec1-1cellscontinuetoprogressthroughmeiosiswithunrepairedDSBs (Lydalletal.1996);assuggestedabove,thepresenceofunrepairedDSBsmayinfluence thedegreeofDNArereplicationinoursystem.However,wedidnotinterrogateTEL1, whichencodesacloserelativeofMec1thatisinvolvedinDNAdamageresponse pathwaysincludingthemeioticrecombinationcheckpointresponse(Greenwelletal. 1995;Morrowetal.1995;Usuietal.2001).LikeMec1,Tel1catalyzesHop1 26 phosphorylation,whichisrequiredforMek1activation(Carballoetal.2008).Itis possiblethatthephenotypicdifferencebetweenSIC1∆PHAdmc1Δmec1Δsml1Δand SIC1∆PHAdmc1Δmek1Δcells(observeddespitethefactthatMek1functions downstreamofMec1)isduetothepresenceofTel1.Alternatively,theremightexist downstreameffectorsofMec1,otherthanMek1,thathaveaninfluencebypromoting DNArereplication. Mec1activationinvolvestheinteractionofitspartnerDdc2withsingle-stranded- DNAboundreplicationproteinA(ZouandElledge2003),whichinthecaseofdmc1Δ cellswouldbeformedreadilyduetothehighlyresectedSpo11-generatedDSBs(Bishop etal.1992).Inturn,Mec1cancatalyzeformationof γ-H2A,whichwefoundcontributed topreventionofDNArereplication,asdidDOT1,encodingtheenzymethatgenerates H3meK79.Becauseγ-H2AwasabundantinSIC1∆PHAcellsthatunderwentDNA rereplication,andH3meK79waslikelytobeabundantaswell(vanLeeuwenetal.2002), thesetwohistonemodificationsarenecessaryforthefullcheckpointresponse,butnot sufficient.Inthemitoticcellcycle,particularlywithrespecttotheG1DNAdamage checkpointresponse,thesemodifications(aswellasRad6-Bre1mediatedhistoneH2B ubiquitylationrequiredforH3meK79generation)areimportantforRad9recruitment andRad53activation(Giannattasioetal.2005;Javaherietal.2006;Hammetetal.2007). InthecheckpointresponsethatpreventsDNArereplication,neitherRAD9norRAD53 appearedtobeinvolved.WhileitistheoreticallypossiblethatRad53canbeactivated byamechanismthatcannotbedetectedbyconventionalmeans(electrophoretic mobilityshiftorinsituautophosphorylation),thispossibilityseemsremote.Infact,our resultsareconsistentwiththefactthattheRad9-Rad53axisisnotinvolvedinthe 27 meioticrecombinationcheckpointthatservestopreventpachyteneexitand progressionthroughthemeioticdivisions(Bishopetal.1992;Lydalletal.1996;Bailis andRoeder2000).Ofparticularinterest,however,isthatresiduesinDbf4,and presumablySld3aswell,thatarephosphorylatedthroughRad53catalysisinthemitotic cellcycletopreventlateoriginfinding(Lopez-Mosquedaetal.2010;Zegermanand Diffley2010),alsofunctioninthemeioticresponsetopreventDNArereplication.By contrasttothemitoticcellcycleobservation(ZegermanandDiffley2010),wefound thatthemcm5-bob1allele,whicheliminatestherequirementfortheDbf4-Cdc7protein kinaseinDNAreplicationinitiation(Hardyetal.1997),didnotsubstituteforthedbf4-4A mutantalleleasitdidinthemitoticcellcyclestudies.Thisdistinctioncouldreflect intrinsicdifferencesinDNAreplicationregulationduringthemitoticcellcycleand meiosis;alternatively,itcouldbeduetotheloweredCDKactivityinourengineeredcells. Takentogether,ourresultssuggestaresponseinwhichaproteinkinasethat recognizesmotifsinasimilarmannertoRad53isactivatedthroughamechanism equivalentinmanywaystotheonethatoperatesinresponsetoDNAdamageduring themitoticcellcycle(seeFigure8).Onelikelycandidateforthiskinaseactivitywouldbe Mek1,givenitsstructuralsimilaritytoRad53anditsmeiosis-specificexpression (RockmillandRoeder1991;LeemandOgawa1992;BailisandRoeder2000).However, ourdataargueagainstthispossibilitybecauseSIC1∆PHAdmc1Δcellsdevoidofboth MEK1andRAD54,designedtoexamineMEK1checkpointfunctionspecifically,didnot displayDNArereplication.Furthermore,whileapeptide-basedinvestigationintothe phosphorylationsitespecificityofyeastproteinkinaseshasplacedRad53andMek1into thesamelargestgroupoffiveclusterings,thetwokinasesarenotcloselyrelatedinthis 28 regardandexhibitaconsiderabledifferenceintheirdegreeofspecificity(Moketal. 2010).Finally,arecentproteomicstudyexploringdmc1∆-dependentMek1activityin meioticcellsdidnotidentifyeitherDbf4orSld3asaMek1substrate(Suhandynataetal. 2016).AnotherkinasewithsomephysicalsimilaritytoRad53isDun1,whichinresponse togenotoxicstressoperatesdownstreamofRad53toregulatenucleotidepoollevels andtranscription(Allenetal.1994;ZhaoandRothstein2002).However,biochemical studiessuggestthatthetwoenzymeshavedifferentsubstratespecificities(Sanchezet al.1997;Uchikietal.2004;Chenetal.2007),andacomprehensiveanalysisof transcriptionalregulationuponDNAdamageindicatesdifferenttargetingbyRad53and Dun1(Jaehnigetal.2013).Therefore,evidencedoesnotexisttoindicatethatMek1or Dun1wouldlikelycatalyzephosphorylationofthesamesitesinSld3andDbf4asRad53, suggestingthatadifferentRad53-likeenzymeispresentinmeiosisthatcaninfluence initiationofDNAreplication. Figure8.AmeioticrecombinationcheckpointresponsecaninhibitDNA rereplication.Thisdiagrambasedonourgeneticanalysisdepictscertainkey proteincomponentsinapathwaythatleadsfromaccumulationofunrepaired DSBstoinhibitionofDNArereplicationintheSIC1∆PHAsystem.Alsoshownisan outlineofthepathwaysthatpreventinter-sisterrepairandmeioticprogression innormalcells.Seetextfordetails. 29 ACKNOWLEDGMENTS WethankStephenKron,MarcoMuzi-Falconi,StephenElledge,PhilipZegerman,and JohnDiffleyforyeaststrains,DougKellogg,ChristopheRedon,andWilliamBonnerfor antibodies,andGrantBrownforplasmidandforprovidinghelpfulcommentsonthe manuscript.TheMicroscopy,Imaging,andCytometryResourcesCoreissupportedin partbyNIHCentergrantP30CA022453totheKarmanosCancerInstituteatWayne StateUniversity,andthePerinatologyResearchBranchoftheNationalInstitutesof ChildHealthandDevelopmentatWayneStateUniversity.NANandLWweresupported inpartbyRuthL.KirschsteinNationalResearchServiceAwardT32-CA009531.This researchwassupportedbyinternalfunds. 30 LITERATURECITED Acosta,I.,D.Ontoso,andP.A.San-Segundo,2011Thebuddingyeastpolo-likekinase Cdc5regulatestheNdt80branchofthemeioticrecombinationcheckpointpathway. 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Zhao,X.,E.G.Muller,andR.Rothstein,1998Asuppressoroftwoessentialcheckpoint genesidentifiesanovelproteinthatnegativelyaffectsdNTPpools.MolCell2 (3):329-340. Zhao,X.,andR.Rothstein,2002TheDun1checkpointkinasephosphorylatesand regulatestheribonucleotidereductaseinhibitorSml1.ProcNatlAcadSciUSA99 (6):3746-3751. Zou,L.,andS.J.Elledge,2003SensingDNAdamagethroughATRIPrecognitionofRPAssDNAcomplexes.Science300(5625):1542-1548. 43 Table1.Yeaststrains. Strain Diploids YGB495 YGB535 YGB604 YGB679 YGB687 YGB689 YGB697 YGB700 YGB703 YGB712 YGB713 YGB721 YGB722 YGB758 YGB759 YGB760 YGB761 YGB785 YGB786 YGB788 YGB789 YGB807 YGB808 YGB809 YGB814 YGB866 YGB867 RelevantGenotype ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3 ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/” mek1∆::kanMX4/” swe1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3swe1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/” swe1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/” pch2∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3pch2∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3hop1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3/”dmc1∆::natR/” hop1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3red1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/” red1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3rad9∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3 dmc1∆::natR/”rad9∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3rad53∆::HIS3/”sml1-1/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/”rad53∆::HIS3/” sml1-1/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3sum1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/” sum1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3mec1∆::LEU2/” sml1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/”mec1∆::LEU2/” sml1∆::kanMX4/” SIC113MYC::kanMX6/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3SIC113myc::kanMX6/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3SIC113myc::kanMX6/” dmc1∆::natR/” rad53∆::HIS3/”sml1-1/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3mcm5-bob1::HIS3/”sld3-38A10his-13myc::kanMX/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/”mcm5bob1::HIS3/”sld3-38A-10his-13myc::kanMX/” Designation SIC1∆PHAa SIC1∆PHAdmc1∆a,b SIC1∆PHAdmc1∆a SIC1∆PHAdmc1∆mek1∆ swe1∆ SIC1∆PHAswe1∆ SIC1∆PHAdmc1∆swe1∆ SIC1∆PHAdmc1∆pch2∆ SIC1∆PHApch2∆ SIC1∆PHAhop1∆ SIC1∆PHAdmc1∆hop1∆ SIC1∆PHAred1∆ SIC1∆PHAdmc1∆red1∆ SIC1∆PHArad9∆ SIC1∆PHAdmc1∆rad9∆ SIC1∆PHArad53∆sml1-1 SIC1∆PHAdmc1∆rad53∆ sml1-1 SIC1∆PHAsum1∆ SIC1∆PHAdmc1∆sum1∆ SIC1∆PHAmec1∆sml1∆ SIC1∆PHAdmc1∆mec1∆ sml1∆ SIC113MYC SIC1∆PHASIC113MYC SIC1∆PHAdmc1∆SIC113MYC rad53∆sml1-1c SIC1∆PHAmcm5-bob1sld3-38A SIC1∆PHAdmc1∆mcm5-bob1 sld3-38A 44 YGB934 YGB938 YGB966 YGB967 YGB1012 YGB1014 YGB1075 YGB1241 YGB1255 Haploid YGB502 ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/”hta1SIC1∆PHAdmc1∆h2a-S129A S129A::his3MX6/”hta2-S129A::TRP1/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/” SIC1∆PHAdmc1∆mek1∆ mek1∆::kanMX4/”rad54∆::TRP1/rad54∆::HIS3 rad54∆ HA ura3-1/ura3-1::HOP1pr-SIC1∆P -URA3dmc1∆::natR/” SIC1∆PHAdmc1∆dot1∆ dot1∆::kanMX6/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/”hta1SIC1∆PHAdmc1∆h2a-S129A S129A::his3MX6/”hta2-S129A::TRP1/”dot1∆::kanMX6/” dot1∆ HA ura3-1/ura3-1::HOP1pr-SIC1∆P -URA3rad54∆::TRP1/rad54∆::HIS3 SIC1∆PHArad54∆ ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3hta1-S129A::his3MX6/”hta2SIC1∆PHAh2a-S129A S129A::TRP1/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3dmc1∆::natR/”dbf4∆::TRP1/” SIC1∆PHAdmc1∆dbf4-4A his3::PDBF4-dbf4-4A::HIS3/”sld3-38A-10his-13myc::kanMX/” sld3-38A HA ura3-1/ura3-1::HOP1pr-SIC1∆P -URA3dmc1∆::natR/” SIC1∆PHAdmc1∆sml1∆ sml1∆::kanMX4/” ura3-1/ura3-1::HOP1pr-SIC1∆PHA-URA3DMC1/dmc1∆::natR SIC1∆PHAdbf4-4Asld3-38A dbf4∆::TRP1/”his3::PDBF4-dbf4-4A::HIS3/”sld3-38A-10his13myc::kanMX/” SIC113MYC::kanMX6 SIC113MYC AllstrainslistedwereconstructedintheW303background(ThomasandRothstein 1989):diploidwildtype=MATa/αade2-1/”ura3-1/”leu2-3,112/”his3-11,15/”trp1-1/” can1-100/”;haploidwildtype=MATaade2-1ura3-1leu2-3,112his3-11,15trp1-1can1100. a Reference(Sawarynskietal.2009). b c ThisSIC1∆PHAdmc1∆strainwasusedonlyfortheexperimentshowninFigureS2. DerivedfromYGB760. 45 Fig. 1 SIC1ΔPHA 0 SIC1ΔPHA dmc1Δ 4 8 24 0 4 8 24 SIC1ΔPHA dmc1Δ mec1Δ sml1Δ 0 4 8 24 SIC1ΔPHA mec1Δ sml1Δ 0 4 8 24 hr HA tub 0 4 8 24 hr 2C 4C 8C 16C DNA re-replication 2C 4C 2C 4C 8C 2C 4C dmc1Δ: unrepaired DSBs dmc1Δ: unrepaired DSBs DNA re-replication Mec1 DNA re-replication 8C DNA re-replication A SIC1ΔPHA dmc1Δ 0 4 B SIC1ΔPHA dmc1Δ red1Δ 8 25 0 4 8 25 hr Fig. 2 SIC1ΔPHA dmc1Δ 0 SIC1ΔPHA dmc1Δ hop1Δ 4 8 26 0 4 8 26 hr HA HA tub tub 0 0 4 4 8 8 25 hr 2C 4C 2C 4C C 8C 26 hr 2C 4C 2C 4C SIC1ΔPHA dmc1Δ SIC1ΔP dmc1Δ mek1Δ 0 0 SIC1ΔPHA dmc1Δ mek1Δ rad54Δ HA SIC1ΔPHA 0 4 8 24 4 8 24 4 8 8C 24 0 4 8 24 hr HA tub ɣ-H2A tub 0 4 8 24 hr 2C 4C 8C 16C 2C 4C 2C 4C 8C 16C 2C 4C Fig. 3 A SIC1ΔPHA dmc1Δ 0 4 8 SIC1ΔPHA sum1Δ 0 24 8 4 SIC1ΔPHA dmc1Δ sum1Δ 0 24 4 8 24 hr HA tub 0 4 8 24 hr 2C 4C 8C 2C 4C 2C 4C C B swe1Δ 0 4 8 24 SIC1ΔPHA swe1Δ 0 4 8 SIC1ΔPHA dmc1Δ 0 24 hr 4 8 24 SIC1ΔPHA dmc1Δ swe1Δ 0 4 HA tub tub 0 4 4 8 8 24 hr 2C 4C 8C 24 hr HA 0 2C 4C 8 24 hr 2C 4C 2C 4C Fig. 4 SIC113MYC 0 2 4 SIC113MYC SIC1ΔPHA 6 8 10 12 24 0 2 4 SIC113MYC SIC1ΔPHA dmc1Δ 6 8 10 12 24 0 2 4 6 8 10 12 24 hr HA tub MYC tub 0 2C 4C 2C 4C 8C 16C 2C 4C 2 4 6 8 10 12 24 hr Fig. 5 SIC1ΔPHA 0 4 SIC1ΔPHA dmc1Δ 8 24 0 4 8 24 hr HA tub 0 4 8 24 hr 2C 4C SIC1ΔPHA dmc1Δ h2a-S129A 0 4 8 24 8C 16C 2C 4C SIC1ΔPHA dmc1Δ dot1Δ 0 4 8 24 SIC1ΔPHA dmc1Δ h2a-S129A dot1Δ 0 4 8 24 hr HA tub 0 4 8 24 hr 2C 4C 8C 2C 4C 8C 2C 4C 8C A SIC1ΔP 0 8 4 HA 12 SIC1ΔP 0 24 4 HA 8 dmc1Δ SIC1ΔPHA dmc1Δ rad9Δ 12 0 24 8 4 12 Fig. 6 24 hr tub HA 0 4 8 12 24 hr 2C 4C 8C 16C 2C B 4C HU SIC1ΔPHA 0 4 8 12 24 SIC1ΔPHA dmc1Δ RAD53 0 0 4 8 12 24 1.5 2C SIC1ΔPHA dmc1Δ rad9Δ 0 4 8 12 24 4C HU RAD53 rad53Δ 0 1.5 0 1.5 hr Rad53 tub 32 P-Rad53 Ponceau Fig. 7 SIC1ΔPHA 0 4 8 24 SIC1ΔPHA dmc1Δ 0 4 8 24 SIC1ΔPHA dmc1Δ dbf4-4A sld3-38A 0 4 8 24 SIC1ΔPHA dmc1Δ mcm5-bob1 sld3-38A 0 4 8 24 hr HA ɣ-H2A tub 0 4 8 24 hr 2C 4C 8C 2C 4C 2C4C 8C 2C 4C Fig. 8 dmc1Δ: unrepaired DSBs 9-1-1 kinase Mec1 Mek1 Dbf4 & Sld3 DNA rereplication inter-sister repair meiotic progression