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1、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:/ *TheXenopusCdc6ProteinIsEssentialfortheInitiationofaSingleRoundofDNAReplicationinCell-FreeExtractsMadefromXenopuseggextractsRxnTime:0-30mi
2、n30-60min60-90min+32PlabeledDNAreplicationproductReplicationinitiationrequiredondouble-strandedDNAsubstrateOnlyelongationrequiredonsingle-strandedDNAsubstrate免疫学信息网http:/ *OrcisrequiredtoloadCDC6;CDC6isrequiredtoloadMCM免疫学信息网http:/ *ImmunostainingforCDC6canpickoutproliferatingcellsinPapsmears免疫学信息网h
3、ttp:/ *Pre-RCformationORC:AsixsubunitproteincomplexwhichhasbeenimplicatedasbeingtheeukaryoticDNAreplicationinitiatorprotein.Subunitsarenamedaccordingtotheirsize,withORC1beingthelargestandORC6beingthesmallestsubunit.YeastORCspecificallybindstoreplicationoriginsinanATPdependentmannerandhasbeenshowntop
4、ossessATPaseactivity.CDC6/Cdc18:Anessentialfactorfortheassemblyofthepre-replicativecomplexesthatco-operateswithCdt1toloadMCM2-7ProteolyzedinyeastsorexportedoutofthenucleusinmammaliancellsattheG1-Stransition.OverexpressionofCdc6inyeastcausesmultipleroundsofDNAreplicationwithoutinterveningmitosis,maki
5、ngitacriticalregulatorofDNAreplication.MCM2-7:Afamilyofsixrelatedproteins(MCM2-MCM7)whichseemtofunctiontogetherinalargemulti-subunitproteincomplex.TheroleofMCMsinDNAreplicationisstillspeculative,rangingfrommediationofprotein-DNAinteractionstoDNAhelicaseactivity.免疫学信息网http:/ *免疫学信息网http:/ *Propidiumi
6、odidestainingNo.ofcellsFluorescenceanalysisandcellsorting(FACS)DNAincellsstainedwithpropidiumiodide.IntensityofstainingineachcellisproportionaltoamountofDNAinthecell.免疫学信息网http:/ *Chromatinimmunoprecipitation(ChIP)testswhetheragivenDNAsequenceisboundbyaproteininvivo免疫学信息网http:/ *免疫学信息网http:/ *MCMhom
7、ologfromM.thermoautotrophicum(anarchaebacterium)hashelicaseactivity.Whatdirection?免疫学信息网http:/ *MtMCMformsdoublehexamers免疫学信息网http:/ *Pre-RCformationORC:AsixsubunitproteincomplexwhichhasbeenimplicatedasbeingtheeukaryoticDNAreplicationinitiatorprotein.Subunitsarenamedaccordingtotheirsize,withORC1bein
8、gthelargestandORC6beingthesmallestsubunit.YeastORCspecificallybindstoreplicationoriginsinanATPdependentmannerandhasbeenshowntopossessATPaseactivity.CDC6/Cdc18:Anessentialfactorfortheassemblyofthepre-replicativecomplexesthatco-operateswithCdt1toloadMCM2-7Proteolyzedinyeastsorexportedoutofthenucleusin
9、mammaliancellsattheG1-Stransition.OverexpressionofCdc6inyeastcausesmultipleroundsofDNAreplicationwithoutinterveningmitosis,makingitacriticalregulatorofDNAreplication.MCM2-7:Afamilyofsixrelatedproteins(MCM2-MCM7)whichseemtofunctiontogetherinalargemulti-subunitproteincomplex.TheroleofMCMsinDNAreplicat
10、ionisstillspeculative,rangingfrommediationofprotein-DNAinteractionstoDNAhelicaseactivity.免疫学信息网http:/ *Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs D/-D/- cells免疫学信息网http:/ *oriPEBNA1EBNA1proteinReplicationfromanorigininEpstein-Barrvirus:aplasmidexpressingEBNA1andcontainingoriPwillreplicateandbemaintainedasanepisomeinmammaliancellsHygromycin-Resistancegene免疫学信息网http:/ *Establishment of drug-resistant coloniesafter transfection of EBV-based plasmid免疫学信息网http:/ *
