Singlemolecule analysis of DNA replication by molecular combing

Efficientduplicationofeukaryoticgenomesreliesonthesequentialactivationofthousandsofreplicationoriginsdistributedalongthechromosomes.Thisprocessfollowsacomplexspatialandtemporalprogram,whichisunderthecontrolofepigeneticmechanismsandistightlylinkedtothefunctionalorganizationofthenucleus(Méchali,2001).RecentevidenceindicatesthatthisreplicationprogramisalsocontrolledbycheckpointkinasesthatmonitorthecorrectexecutionofSphase,suchasATRinhumanandMec1inbuddingyeast(TourriereandPasero,2007).Overthelasttwentyyears,awidevarietyofmethodshasbeendevelopedtomapreplicationoriginsineukaryoticcells(DePamphilis,1997).ThesetechniqueshaveshownthateukaryoticgenomescontainanexcessofpotentialreplicationoriginsandthatonlyafractionoftheseoriginsfirewithinagivenSphase.However,thedynamicsofDNAreplicationremainspoorlydefinedatthelevelofindividualchromosomes,essentiallybecausebiochemicalassaysprovideaveragedreplicationprofilesforapopulationofcells.

Therecentdevelopmentofsingle-moleculeassayshasshednewlightonthedynamicsofDNAreplicationatthelevelofindividualchromosomes.ThesetechniqueshavebeensuccessfullyusedtomonitorDNAreplicationinavarietyoforganisms,includingbacteria,yeasts,xenopusandmammals(Anglanaetal.,2003;Breieretal.,2005;Herricketal.,2000;JacksonandPombo,1998;Lemaitreetal.,2005;Paseroetal.,2002;Pateletal.,2006).Here,wedescribetheanalysisofDNAreplicationinS.cerevisiaebyDNAcombing,oneofthemostwidelyusedsingle-moleculeassay(Bensimonetal.,1994;Michaletetal.,1997).

Inthisassay,replicationoriginsarefirstlabeledwithbromodeoxyuridine(BrdU)inearlySphase.ChromosomalDNAisthenpurifiedinagaroseplugsandstretchedonsilanizedcoverslips.Thisproceduregenerateslong,parallelDNAfibers,withauniformextensionof2kb/µm.Newly-replicatedDNAisthendetectedwithamonoclonalantibodydirectedagainstBrdUandDNAfibersarecounterstainedwithanantibodyagainstsingle-strandedDNA.ReplicatingDNAfibersarerevealedwithfluorescentsecondaryantibodiesandareimagedwithanepifluorescencemicroscopecoupledtoaCCDcamera.RepresentativeexamplesoflargeDNAfibers(>400kb)areshowninFigure1.Fromtheseimages,alargenumberofreplicationparameterscanbederived,suchastheratesofinitiationandelongationandthepercentageofsubstitutionforindividualDNAfibers.Whencombinedwithfluorescenceinsituhybridization,thistechniqueallowsaprecisemappingofactiveoriginsalongachromosomeofinterest(Anglanaetal.,2003;Lebofskyetal.,2006;Paseroetal.,2002).DNAcombingcanalsobeusedtomonitorforkrecoveryafterareplicationstress,usingacombinationofhalogenatednucleotides(CldU/IdU)thatcanbedistinguishedwithspecificanti-BrdUantibodies(Lukeetal.,2006;Tourriereetal.,2005).

TheprotocoldescribedbelowismeanttodetectBrdUincorporationinyeastcellsarrestedinearlySphasewithhydroxyurea(HU).ItcanbeeasilyadaptedtomonitorongoingDNAreplicationinasynchronousyeastculturesortoanalyzeDNAreplicationinmammaliancells.SpecificdetailsregardingthepreparationofBrdU-labeledgenomicDNAfrommammaliancellsareavailableuponrequest.

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BrdUlabelingofHU-arrestedcells

YeastcellsarenormallyunabletoincorporateBrdUbecausetheylackthymidinekinaseactivityandsynthesizedTMPdenovo.Wethereforeusegenetically-modifiedstrainscontainingsevencopiesoftheHerpessimplexTKgene(Lengronneetal.,2001).SinceBrdUaccumulatesinyeastcellsbypassivediffusion,wealsotransformcellswithaCEN-basedplasmidbearingthehumannucleosidetransporterhENT1(giftofGrantBrown,Toronto)toaccelerateBrdUuptake.AlternativestrategiestofacilitateBrdUincorporationinyeasthavebeendeveloped(Vernisetal.,2003;ViggianiandAparicio,2006)butthecombinationof7integratedcopiesofHSV-TKplushENT1onaplasmidremainsthemostefficientoptioninourhands.

1. Growcellsovernightat25°Ctoadensityof5.10⁶cells/mlin100mlofcompletesyntheticmedium.
2. Addα-factor(1µg/ml)foratleast2.5hourstoarrestcellsinG₁.Addaseconddoseofα-factorpartwaythroughtheincubationtoensurethatcellsdonotescapetheG₁block.
3. AddBrdUtoafinalconcentrationof400µg/ml(or40µg/mlifcellsexpresshENT1),atleast15minutesbeforereleasingcellsintoSphase(seenote1).
4. AddHUtoafinalconcentrationof200mM(15mg/ml)andreleasecellsfromα-factorwiththeadditionof50µg/mlPronase(Calbiochem).AdjustthepHofthemediumto7.0withphosphatebuffer(seenote2).
5. After90minutes,addsodiumazideintheculturetoafinalconcentrationof0.1%andcollectcellsbycentrifugation.
6. Checkcellsunderthemicroscope.Morethan90%ofthecellsshoulddisplaysmallbuds,whichareindicativeofentryintoSphase.

PreparationofgenomicDNAplugs

GenomicDNAispreparedinlowmeltingpoint(LMP)agaroseplugsinordertopreventitsmechanicalshearing.Plugsarestableforseveralmonthsat4°C.

1. Washcellswith10mMTris-HCl,50mMEDTA,pH8.0anddeterminecellconcentrationwithacellcounter.
2. ResuspendedcellsinprewarmedZymolyasebuffer(42°C)toafinalconcentrationof1.10⁹cells/ml.
3. AddanequalvolumeofmoltenLMPagarose(42°C)andprepareagaroseplugsusingaplugmold(GEAmersham).Eachplug(90µl)shouldcontain5.10⁷cellsorapproximately850ngofgenomicDNA.
4. Letagaroseplugssolidifyfor30minutesat4°C.
5. UseaPasteurpipetterubberbulbtoejectplugsdirectlyinto12mlround-bottomtubescontainingZymolyasebuffer(0.4mlperplug).Incubateovernightat37°C.
6. ReplaceZymolyasebufferwithProteinaseKbuffer(0.4mlperplug)andincubatefor48hoursat50°C.
7. Wash5x10minin10mlof10mMTris,50mMEDTAandstoreplugsat4°C.

DNAcombing

DNAcombingisperformedonsilanizedcoverslipsessentiallyasdescribedpreviouslybytheBensimonlab(Michaletetal.,1997).

1. StainDNAplugsfor30minwith1.5µlYOYO-1(MolecularProbes)in100µlTE.
2. Washthoroughlyin10mlTE(3x5min)andtransfereachpluginaround-bottompolycarbonatetubecontaining5mlof50mMMESpH5.7
3. Melttheagaroseplugsfor15minat67°Cwithaheatingblock(seenote3).
4. Letthesolutioncooldownto42°Cbeforeaddingβ-agarase(3units,NewEnglandBiolabs)andincubateovernight.
5. Incubateat65°Cfor10minutesandstoreatroomtemperatureuntiluse.
6. CarefullypourtheDNAsolutionintoa2mlTeflonreservoir.Savetherestforfurtheruse.
7. InsertasilanizedcoverslipintotheDNAsolutionandincubatefor5minutesatroomtemperature.Removecarefullythecoverslipfromthereservoiratthespeedof300µm/s(seenote4).Repeatwithanothercoverslipasmanytimesasneeded.
8. CheckDNAfibersunderthemicroscopeusinga40xobjectiveandaFITCfilterblock.Tothisaim,attachthecoversliptoametalholderwithaholeinthemiddle,putadropofimmersionoildirectlyonthecoverslipandvisualizeDNAfibersonthebottomsideofthecoverslip.
9. PlacethecoversliponasheetofWhatmanpaperandbakefor2hat60°CtocrosslinkDNAtocoverslips.
10. Stickcoverslipsonglassslideswithcyanoacrylateglue.Labelslideswithadiamondtipengravingpenandstoreat-20°Cuntiluse.

Immunodetection

ThefollowingprotocolisfortheimmunodetectionofBrdU-substitutednascentDNAandparentalDNAfibers,asillustratedinfigure1a.OthercombinationsofprimaryantibodiescanbeusedtovisualizepulsesofCldUandIdUortodetectCldU,IdUandDNAfiberssimultaneously.Fluorescenceinsituhybridization(FISH)canalsobeperformedifneeded(seeoptionalprotocolbelow).

1. Dehydrateslidesfor5mininsuccessivebathsof70%,90%and100%EtOH.
2. Incubatefor25minin1MNaOH.
3. WashextensivelywithPBSpH7.4toneutralizeNaOH(5washesof1minuteeach).
4. PerformFISHatthisstep(seenextsection),otherwiseproceedwithstep5.
5. Incubatefor15mininPBS/Tcontaining1%BSA.
6. Add18µlofPBS/Tcontainingprimaryantibodiesandcoverwithacoverslip.Incubatefor45minat37°Cinahumidchamber.
7. Wash5x2minutestimeswithPBS/T(seenote5).
8. Detectwithsecondaryantibodies(30minat37°Cinhumidchamber)
9. Wash5x3minuteswithPBS/T.
10. Dryslidesandmountwith20µlofProlongGoldAntifadereagent(MolecularProbes).Letmountingreagentpolymerasefor2hoursatroomtemperaturebeforeproceedingwithmicroscopy.Mountedcoverslipsarestableformonthsat-20°C.

Fluorescenceinsituhybridization(optional)

1. AmplifyprobeDNA(1to3kb)byPCRandpurifyproductsonQiagencolumns.
2. LabelprobewithDIG-dUTPbyrandomprimingandeliminatefreelabelonaG50column.
3. Adda20-foldexcessofcompetitorDNA(salmonspermDNA)andEtOHprecipitate.
4. ResuspendprobeinProbemixtoafinalconcentrationof10to50ng/µlanddenaturefor5minat65°C.
5. DiluteProbemixinHybridizationmixtoafinalconcentrationof2ng/µl.
6. Add20to50µlofhybridizationmixtureperslide,sealwithanadhesivehybridizationchamberandincubateat37°Cforatleast5hours.
7. Wash3x5minutesin2xSSC,50%formamide.
8. Wash3x5minutesin2xSSC.
9. Wash3x5minutesinPBS/T.
10. Proceedwithstep5of“Immunodetection”section.Useamouseanti-DIGantibody(Roche)insteadoftheanti-DNAantibodytovisualizeFISHprobes.

Imageacquisitionandanalysis

ImageacquisitionisperformedwithafullymotorizedLeicaDM6000BmicroscopeequippedwithaCoolSNAPHQCCDcameraandcontrolledwithMetaMorph(RoperScientific).OnimagesacquiredwiththisCCDcameraanda40xobjective,1pixel=340bp.BrdUtracksandDNAfibersaremeasuredmanuallywithanofflineversionofMetaMorphanddataaretransferredtoanExcelspreadsheet(Microsoft).StatisticalanalysisofBrdUtracklengthandinter-origindistancesisperformedwithPrism5.0(GraphPad),asdescribedinfigure1.

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1. BrdUisdissolvedin10%DMSOataconcentrationof5mg/mlandstoredat-20°C.
2. Weusuallysave20mloftheculturetomonitorcellcycleprogressionbyFACSintheabsenceofHU.CellsshouldenterSphase20-30minaftertheadditionofPronase.
3. Fromthisstep,theDNAsolutionshouldbemanipulatedverygently.
4. CombingdevicesarenolongerdistributedbyPasteurInstruments.ContactAaronBensimon(a.bensimon@genomicvision.com)forcombingmachinesandsilanizedcoverslips.Protocolsforgas-phasesilanisationofcoverslipsandinstructionsfortheassemblyofcustomcombingdevicesareavailableuponrequest.
5. DiptheslideinabeakercontainingPBS/TinordertoremovetheuppercoverslipwithoutdamagingtheDNAfibers.

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Figure1:AnalysisofDNAreplicationprofilesinbuddingyeastbyDNAcombing.(A)ImagesofrepresentativeDNAfibersfromdifferentfieldsofviewwereprocessedwithAdobePhotoshoptoeliminatebackgroundsignalsandarbitrarilyalignedtostresstheclusteringofearly-firingorigins.Green:BrdU.Red:DNA.Scalebaris100kb.(B)BrdUtracks(greenchannelonly).(C)DistributionofBrdUtracklengthinHU-arrestedwildtypecells.(D)Distributionofinter-origindistances(IODs)inHU-arrestedwildtypecells.BrdUtracklengthandIODsweremeasuredwithMetaMorph(RoperScientific)andfrequencydistributionswerecalculatedwithPrism5.0(GraphPad).Medianvaluesareindicatedinkb.(E)DistributionofactiveoriginsalongtheyeastrDNAarray.EachrDNAunitcontainsapotentialreplicationoriginbutonly20%oftheseoriginsfireeverycellcycle.CombinedFISH(reddots)andBrdUdetection(greentracks)showthatactiveoriginsformclustersof~3adjacentunitsseparatedfromeachotherwithlargesilentregions.ThisreplicationpatternisimposedatleastinpartbythehistonedeacetylaseSir2(Paseroetal,2002).