复制第五讲复制第五讲G2MORCMCMMCMCDC6 +Cdt1Replication Machinery“Pre-Replicative Complex”Replication complexes during the cell cycleORCCDC6+Cdt1Slate G1early G1免疫学信息网http:/www.immuneweb.com *TheXenopusCdc6ProteinIsEssentialfortheInitiationofaSingleRoundofDNAReplicationinCell-FreeExtractsMadefromXenopuseggextractsRxnTime:0-30min30-60min60-90min+32PlabeledDNAreplicationproductReplicationinitiationrequiredondouble-strandedDNAsubstrateOnlyelongationrequiredonsingle-strandedDNAsubstrate免疫学信息网http:/www.immuneweb.com *OrcisrequiredtoloadCDC6;CDC6isrequiredtoloadMCM免疫学信息网http:/www.immuneweb.com *ImmunostainingforCDC6canpickoutproliferatingcellsinPapsmears免疫学信息网http:/www.immuneweb.com *Pre-RCformationORC:AsixsubunitproteincomplexwhichhasbeenimplicatedasbeingtheeukaryoticDNAreplicationinitiatorprotein.Subunitsarenamedaccordingtotheirsize,withORC1beingthelargestandORC6beingthesmallestsubunit.YeastORCspecificallybindstoreplicationoriginsinanATPdependentmannerandhasbeenshowntopossessATPaseactivity.CDC6/Cdc18:Anessentialfactorfortheassemblyofthepre-replicativecomplexesthatco-operateswithCdt1toloadMCM2-7ProteolyzedinyeastsorexportedoutofthenucleusinmammaliancellsattheG1-Stransition.OverexpressionofCdc6inyeastcausesmultipleroundsofDNAreplicationwithoutinterveningmitosis,makingitacriticalregulatorofDNAreplication.MCM2-7:Afamilyofsixrelatedproteins(MCM2-MCM7)whichseemtofunctiontogetherinalargemulti-subunitproteincomplex.TheroleofMCMsinDNAreplicationisstillspeculative,rangingfrommediationofprotein-DNAinteractionstoDNAhelicaseactivity.免疫学信息网http:/www.immuneweb.com *免疫学信息网http:/www.immuneweb.com *PropidiumiodidestainingNo.ofcellsFluorescenceanalysisandcellsorting(FACS)DNAincellsstainedwithpropidiumiodide.IntensityofstainingineachcellisproportionaltoamountofDNAinthecell.免疫学信息网http:/www.immuneweb.com *Chromatinimmunoprecipitation(ChIP)testswhetheragivenDNAsequenceisboundbyaproteininvivo免疫学信息网http:/www.immuneweb.com *免疫学信息网http:/www.immuneweb.com *MCMhomologfromM.thermoautotrophicum(anarchaebacterium)hashelicaseactivity.Whatdirection?免疫学信息网http:/www.immuneweb.com *MtMCMformsdoublehexamers免疫学信息网http:/www.immuneweb.com *Pre-RCformationORC:AsixsubunitproteincomplexwhichhasbeenimplicatedasbeingtheeukaryoticDNAreplicationinitiatorprotein.Subunitsarenamedaccordingtotheirsize,withORC1beingthelargestandORC6beingthesmallestsubunit.YeastORCspecificallybindstoreplicationoriginsinanATPdependentmannerandhasbeenshowntopossessATPaseactivity.CDC6/Cdc18:Anessentialfactorfortheassemblyofthepre-replicativecomplexesthatco-operateswithCdt1toloadMCM2-7ProteolyzedinyeastsorexportedoutofthenucleusinmammaliancellsattheG1-Stransition.OverexpressionofCdc6inyeastcausesmultipleroundsofDNAreplicationwithoutinterveningmitosis,makingitacriticalregulatorofDNAreplication.MCM2-7:Afamilyofsixrelatedproteins(MCM2-MCM7)whichseemtofunctiontogetherinalargemulti-subunitproteincomplex.TheroleofMCMsinDNAreplicationisstillspeculative,rangingfrommediationofprotein-DNAinteractionstoDNAhelicaseactivity.免疫学信息网http:/www.immuneweb.com *Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs D/-D/- cells免疫学信息网http:/www.immuneweb.com *oriPEBNA1EBNA1proteinReplicationfromanorigininEpstein-Barrvirus:aplasmidexpressingEBNA1andcontainingoriPwillreplicateandbemaintainedasanepisomeinmammaliancellsHygromycin-Resistancegene免疫学信息网http:/www.immuneweb.com *Establishment of drug-resistant coloniesafter transfection of EBV-based plasmid免疫学信息网http:/www.immuneweb.com *