斯坦福大学分子生物学课件-Replication3A.ppt

斯坦福大学分子生物学课件-Replication3A.ppt

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Formationofpre-ReplicativecomplexG2MORCMCMMCMCDC6+Cdt1ReplicationMachinery“Pre-ReplicativeComplex”ReplicationcomplexesduringthecellcycleORCCDC6+Cdt1SlateG1earlyG1EukaryoticinitiationcomplexORC:

AsixsubunitproteincomplexwhichhasbeenimplicatedasbeingtheeukaryoticDNAreplicationinitiatorprotein.Subunitsarenamedaccordingtotheirsize,withORC1beingthelargestandORC6beingthesmallestsubunit.YeastORCspecificallybindstoreplicationoriginsinanATPdependentmannerandhasbeenshowntopossessATPaseactivity.CDC6/Cdc18:

Anessentialfactorfortheassemblyofthepre-replicativecomplexesthatco-operateswithCdt1toloadMCM2-7ProteolyzedinyeastsorexportedoutofthenucleusinmammaliancellsattheG1-Stransition.OverexpressionofCdc6inyeastcausesmultipleroundsofDNAreplicationwithoutinterveningmitosis,makingitacriticalregulatorofDNAreplication.MCM2-7:

Afamilyofsixrelatedproteins（MCM2-MCM7）whichseemtofunctiontogetherinalargemulti-subunitproteincomplex.TheroleofMCMsinDNAreplicationisstillspeculative,rangingfrommediationofprotein-DNAinteractionstoDNAhelicaseactivity.A/TDUEOREAuxiliaryelementsAuxiliaryelementsOriginrecognitionproteinsTranscriptionFactorsTranscriptionFactorsLeadingstrLeadingstrLaggingstrLaggingstrCoreoriginOriginsaremulti-partiteTheStructureofARS1（S.cerevisiae）B1ACSB2B3ABF1ORC“Post-RC”protection“Pre-RC”protection134564122ORCusesdifferentstrategiesforbindingDNAindifferentspeciesPre-RCformationORC:

AsixsubunitproteincomplexwhichhasbeenimplicatedasbeingtheeukaryoticDNAreplicationinitiatorprotein.Subunitsarenamedaccordingtotheirsize,withORC1beingthelargestandORC6beingthesmallestsubunit.YeastORCspecificallybindstoreplicationoriginsinanATPdependentmannerandhasbeenshowntopossessATPaseactivity.CDC6/Cdc18:

Anessentialfactorfortheassemblyofthepre-replicativecomplexesthatco-operateswithCdt1toloadMCM2-7ProteolyzedinyeastsorexportedoutofthenucleusinmammaliancellsattheG1-Stransition.OverexpressionofCdc6inyeastcausesmultipleroundsofDNAreplicationwithoutinterveningmitosis,makingitacriticalregulatorofDNAreplication.MCM2-7:

Afamilyofsixrelatedproteins（MCM2-MCM7）whichseemtofunctiontogetherinalargemulti-subunitproteincomplex.TheroleofMCMsinDNAreplicationisstillspeculative,rangingfrommediationofprotein-DNAinteractionstoDNAhelicaseactivity.TheXenopusCdc6ProteinIsEssentialfortheInitiationofaSingleRoundofDNAReplicationinCell-FreeExtractsMadefromXenopuseggextractsRxnTime:

0-30min30-60min60-90min+32PlabeledDNAreplicationproductReplicationinitiationrequiredondouble-strandedDNAsubstrateOnlyelongationrequiredonsingle-strandedDNAsubstrateOrcisrequiredtoloadCDC6;CDC6isrequiredtoloadMCMImmunostainingforCDC6canpickoutproliferatingcellsinPapsmearsPre-RCformationORC:

AsixsubunitproteincomplexwhichhasbeenimplicatedasbeingtheeukaryoticDNAreplicationinitiatorprotein.Subunitsarenamedaccordingtotheirsize,withORC1beingthelargestandORC6beingthesmallestsubunit.YeastORCspecificallybindstoreplicationoriginsinanATPdependentmannerandhasbeenshowntopossessATPaseactivity.CDC6/Cdc18:

Anessentialfactorfortheassemblyofthepre-replicativecomplexesthatco-operateswithCdt1toloadMCM2-7ProteolyzedinyeastsorexportedoutofthenucleusinmammaliancellsattheG1-Stransition.OverexpressionofCdc6inyeastcausesmultipleroundsofDNAreplicationwithoutinterveningmitosis,makingitacriticalregulatorofDNAreplication.MCM2-7:

Afamilyofsixrelatedproteins（MCM2-MCM7）whichseemtofunctiontogetherinalargemulti-subunitproteincomplex.TheroleofMCMsinDNAreplicationisstillspeculative,rangingfrommediationofprotein-DNAinteractionstoDNAhelicaseactivity.PropidiumiodidestainingNo.ofcellsFluorescenceanalysisandcellsorting（FACS）DNAincellsstainedwithpropidiumiodide.IntensityofstainingineachcellisproportionaltoamountofDNAinthecell.Chromatinimmunoprecipitation（ChIP）testswhetheragivenDNAsequenceisboundbyaproteininvivoMCMhomologfromM.thermoautotrophicum（anarchaebacterium）hashelicaseactivity.Whatdirection?

MtMCMformsdoublehexamersPre-RCformationORC:

AsixsubunitproteincomplexwhichhasbeenimplicatedasbeingtheeukaryoticDNAreplicationinitiatorprotein.Subunitsarenamedaccordingtotheirsize,withORC1beingthelargestandORC6beingthesmallestsubunit.YeastORCspecificallybindstoreplicationoriginsinanATPdependentmannerandhasbeenshowntopossessATPaseactivity.CDC6/Cdc18:

Anessentialfactorfortheassemblyofthepre-replicativecomplexesthatco-operateswithCdt1toloadMCM2-7ProteolyzedinyeastsorexportedoutofthenucleusinmammaliancellsattheG1-Stransition.OverexpressionofCdc6inyeastcausesmultipleroundsofDNAreplicationwithoutinterveningmitosis,makingitacriticalregulatorofDNAreplication.MCM2-7:

Afamilyofsixrelatedproteins（MCM2-MCM7）whichseemtofunctiontogetherinalargemulti-subunitproteincomplex.TheroleofMCMsinDNAreplicationisstillspeculative,rangingfrommediationofprotein-DNAinteractionstoDNAhelicaseactivity.Mammaliancellscansurvivewith0.1xthenormalORC2level:

WTvsD/-D/-cellsoriPEBNA1EBNA1proteinReplicationfromanorigininEpstein-Barrvirus:

aplasmidexpressingEBNA1andcontainingoriPwillreplicateandbemaintainedasanepisomeinmammaliancellsHygromycin-ResistancegeneEstablishmentofd