KatherineFriedman Friedman,K.L.,M.K.Raghuraman,W.L.Fangman,andB.J.Brewer(1995)AnalysisofthetemporalprogramofreplicationinitiationinyeastchromosomesJ.CellSci.Suppl.19:51-58. TheE.colidammethylaseaddsmethylgroupstoadenineinthesequence5"GATC3"(MarinusandMorris,1973;GeierandModrich,1979).Thisenzymemethylatesbothunmethylatedandhemi-methylatedsites.InyeastDNA,methylatedbasesareundetectable,unliketheDNAofmosthighereukaryotes(Hattmanetal.,1978).However,thedammethylaseproteincanbeexpressedatmoderatelevelsinyeastwithfewobviouseffectsoncellviABIlityorgrowthrate(Brooksetal.,1983;HoekstraandMalone,1985).ExpressionoftheproteinresultsinmoderatelevelsofDNAmethylation.TheaccessibilityofdifferentGATCsitestomethylationisvariable,presumablyduetodifferencesinchromatinstructurethataffecttheaccessoftheenzymetoDNA(Gottschling,1992;SinghandKlar,1992;Wrightetal.,1992;KladdeandSimpson,1994). E.coliDNA,thoughnormallyfullymethylated,undergoesatransientperiodofhemimethylationimmediatelyfollowingpassageofthereplicationfork(reviewedinBarrasandMarinus,1989).Thenewlysynthesizedstrandisnotinitiallymethylated,butacquiresmethylationafterdammethylasecontactsthenewlyreplicatedDNAsequence.Methylatedandhemi-methylatedsequencescanbedistinguishedbytheirdifferentialsensitivitytocleavagebyDpnI(GeierandModrich,1979).FullymethylatedDNAiscleavedatGATCsitesandhemi-methylatedorunmethylatedDNAisnot(Figure1).Therefore,thetimeatwhichE.colisequencesbecomeresistanttocleavagebyDpnIcorrelateswiththeirtimeofreplication.AsimilarlogiccanbeappliedtoreplicationofyeastDNAinastrainexpressingthedammethylase.Again,replicationforkpassageresultsinhemi-methylationoftheDNA,detectableasresistancetocleavagebyDpnI(Figure1). InordertoconstructastraincontainingconstitutivelymethylatedDNA,theE.colidammethylasegeneanditsendogenouspromoterwereexcisedfromplasmidpMFH1(HoekstraandMalone,1985)withHindIIIandPvuIIandinsertedintotheHindIII-SmaIsitesofthepolylinkerinpRS305(SikorskiandHieter,1989),aLeu+yeastintegratingvector.Theresultingplasmid(pRS305-DAM)waspartiallydigestedwithClaIandthelinearizedplasmidwasusedtotransformyeaststrainRM14-3a(MATacdc7-1bar1ura3-52trp1-289leu2-3,112his6).CleavageoftransformantDNAwithDpnIwasassessedbySouthernhybridization,andthetransformantexhibitingthehighestlevelofDpnIcleavagewasselected(RM14-3aMeth5strain). 1.Grow~330mlofRM14-3aexpressingdammethylase(RM14-3aMeth5)toanOD660ofabout0.3(3.85x106cells/ml)at23oC.Arrestwith200nMalphafactorfor1-1.25doublingtimes,untilthepercentbuddedcellsstopsincreasing(usually?5%).(RM14-3aisbar1,sothealphafactorconcentrationrequiredislow). 2.Transfercellsto37oCandwaitforcultureitselftoreach37oC.AddPronase(20microgram/mlfinalconcentration--dissolvePronasein5mlminimalmedium).WeusePronasefromCalbiochem. 3.Holdat37oCfor~120minutes(until%buddedcellsreaches95-98%).Removetwosamplesfor0timepoint,thenswirltheflaskinicewaterforoneminuteandreturnto23oC.Iusuallycollectsampleseveryfiveminutesfor55minutes,thencollect65and80minutetimepoints. 4.Collectingsamples:Freeze8mlof0.1%Na-azide,0.2MEDTAin"50ml"Oakridge-typeplasticscrewcapcentrifugetubes(freezetheseslantedat-20oCtoincreasesurfacearea).Duringtheexperiment,mix20mlculturewitha1/20volumeof10%Na-azideina35mlCorextube(onice),immediatelytransfertothetubeoffrozenEDTA/azide,andshakethetubetochillthesampleandmeltthefrozenEDTA.Spinthecellsdowninachilledcentrifuge,washwith1mlcoldwaterinEppendorftubes,andfreezethepelletsat-20oC. 5.ExtracttheDNAbysmashandgrabmethod. 6.Restrictiondigest: Figure2AandBshowatypicalresultforARS1andR11. Theblotsarequantitatedbyphosphorimager.TheextentofDpnIcleavageisexpressedasthefractionoftotalsignalinthelanethatisattributabletothefull-lengthEcoRIfragment.ThisfractionshouldincreaseasthefragmentisreplicatedbecausetheDNAbecomestransientlyresistanttoDpnIcleavage.Thefractionofsignalinthefull-lengthEcoRIfragmentisthennormalizedtothelargestvalueandplottedversusthetimeafterreleasefromthecdc7block(seeFigure2C). Barras,F.,andM.G.Marinus.1989.ThegreatGATC:DNAmethylationinE.coli.TrendsGenet.5:139-143. Brooks,J.E.,R.M.Blumenthal,andT.R.Gingeras.1983.TheisolationandcharacterizationoftheEscherichiacoliDNAadeninemethylase(dam)gene.NucleicAcidsRes.11:837-851. Geier,G.E.,andP.Modrich.1979.RecognitionsequenceofthedammethylaseofEscherichiacoliK12andmodeofcleavageofDpnIendonuclease.J.Biol.Chem.254:1408-1413. Gottschling,D.E.1992.Telomere-proximalDNAinSaccharomycescerevisiaeisrefractorytomethyltransferaseactivityinvivo.Proc.Natl.Acad.Sci.USA89:4062-4065. Hattman,S.,C.Kenny,L.Berger,andK.Pratt.1978.ComparativestudyofDNAmethylationinthreeunicellulareucaryotes.J.Bacteriol.135:1156-1157. Hoekstra,M.F.,andR.E.Malone.1985.ExpressionoftheEscherichiacolidammethylaseinSaccharomycescerevisiae:Effectofinvivoadeninemethylationongeneticrecombinationandmutation.Mol.Cell.Biol.5:610-618. Kladde,M.P.,andR.T.Simpson.1994.Positionednucleosomesinhibitdammethylationinvivo.Proc.Natl.Acad.Sci.Usa91:1361-1365. Marinus,M.G.,andN.R.Morris.1973.IsolationofdeoxyribonucleicacidmethylasemutantsofEscherichiacoliK-12.J.Bacteriol.114:1143-1150. Sikorski,R.S.,andP.Hieter.1989.AsystemofshuttlevectorsandyeasthoststrainsdesignedforefficientmanipulationofDNAinSaccharomycescerevisiae.Genetics122:19-27. Singh,J.,andA.J.Klar.1992.ActivegenesinbuddingyeastdisplayenhancedinvivoaccessibilitytoforeignDNAmethylases:anovelinvivoprobeforchromatinstructureofyeast.GenesDev.6:186-196. Wright,J.H.,D.E.Gottschling,andV.A.Zakian.1992.Saccharomycestelomeresassumeanon-nucleosomalchromatinstructure.GenesDev.6:197-210.
Reference:Rationaleofmethylationtimingapproach
Strainconstruction
Protocolformethylationtiming
Quantitation
References