Download Prevention of DNA Rereplication Through a Meiotic Recombination

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.
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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
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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
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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
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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.
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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
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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
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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.
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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).
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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,
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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.
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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
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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