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1、复制第三讲复制第三讲免疫学信息网http:/1) In synchronously replicating cells, density transfer experiments can reveal which sequences replicate earliest. What is the direction of fork movement here?Time% of probe in HL DNA0100ABCCBAGenes on achromosome免疫学信息网http:/Linker scanning mutations in yeast ARS1% URA+ colonie
2、sExamples of mutantsAB1B2B3WT-ARS1免疫学信息网http:/免疫学信息网http:/OriC in E. coli chromosomal DNA免疫学信息网http:/Initiation of DNA replication in E. coli免疫学信息网http:/ Two primosomes for E. coli chromosomal replicationSite of primosome assemblyoriC, A-siten-pas, D-loop, R-loopRecognitionDnaA proteinPriA proteinAu
3、xilliary proteins for(HU for oriC)PriBloading helicase PriCDnaTHelicaseDnaB/DnaCPrimingPrimaseDnaA and oriC independent replication initiation in E. colifrom hairpins, D-loops and R loops免疫学信息网http:/SV40: a polyomavirus that is very useful forstudying eukaryotic replication enzymes免疫学信息网http:/Early
4、genes : proteins essential for viral DNA replication免疫学信息网http:/T antigen binding sites cluster around the ori免疫学信息网http:/* 98% nuclear: NLS * origin-specific DNA binding * DNA independent ATPase * ATP dependent DNA helicase * Binds to DNA polymerases, AP2, p53, Rb * Undergoes phosphorylation etc.Pr
5、operties of SV40 T antigen免疫学信息网http:/COS cells (infected with SV40)(expresses T Ag) + plasmid + dNTP + rNTP+ 32PdATP37o C, 60 minAnalyze products by electrophoresis and autoradiographySV40 oriSV40 - +ori - +Li & KellySV40 DNA replicationIn vitro.PNAS (1984) 81:6973What else could they doto validate
6、 their system?免疫学信息网http:/Fractionation to homogeneityCOS cells (infected with SV40)(expresses T Ag)T Antigen frombaculovirus expression systemHuman HeLa or 293 cellscell lysatePC0.2 M KCl0.66 M KCl QS0.2 M KCl0.4 M KCl (PCNA)ssDNA0.6 M KCl1 M KClRPA+(RF-C, pol + pol )免疫学信息网http:/ProkaryoticFunctionEukaryoticSSBss DNA coatingRPAPRIMASERNA primer synth.Primase subunitof DNA POL ,5-3 polymeraseDNA POL ,DNA POL POL III CORE 3-5 exonucleaseDNA POL 5-3 exonucleaseFen1 COMPLEXATP dependentclamploaderRF-C CLAMPprocessivity factorPCNALIGASEseal nicksLig1 DnaBHelicase?ELONGATION FACTORS ARE CONSERVED