复制第五讲.ppt

免疫学信息网http:/,Formation of pre-Replicative complex,免疫学信息网http:/,G2,M,CDC6+Cdt1,“Pre-Replicative Complex”,Replication complexes during the cell cycle,S,late G1,early G1,免疫学信息网http:/,Eukaryotic initiation complex,ORC:A six subunit protein complex which has been implicated as being the eukaryotic DNA replication initiator protein.Subunits are named according to their size,with ORC1 being the largest and ORC6 being the smallest subunit.Yeast ORC specifically binds to replication origins in an ATP dependent manner and has been shown to possess ATPase activity.CDC6/Cdc18:An essential factor for the assembly of the pre-replicative complexes that co-operates with Cdt1 to load MCM2-7Proteolyzed in yeasts or exported out of the nucleus in mammalian cells at the G1-S transition.Overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis,making it a critical regulator of DNA replication.MCM2-7:A family of six related proteins(MCM2-MCM7)which seem to function together in a large multi-subunit protein complex.The role of MCMs in DNA replication is still speculative,ranging from mediation of protein-DNA interactions to DNA helicase activity.,免疫学信息网http:/,Leading str,Leading str,Lagging str,Lagging str,Core origin,Origins are multi-partite,免疫学信息网http:/,The Structure of ARS1(S.cerevisiae),B,1,ACS,B,2,B,3,ORC,免疫学信息网http:/,免疫学信息网http:/,ORC uses different strategies for binding DNAin different species,免疫学信息网http:/,Pre-RC formation,ORC:A six subunit protein complex which has been implicated as being the eukaryotic DNA replication initiator protein.Subunits are named according to their size,with ORC1 being the largest and ORC6 being the smallest subunit.Yeast ORC specifically binds to replication origins in an ATP dependent manner and has been shown to possess ATPase activity.CDC6/Cdc18:An essential factor for the assembly of the pre-replicative complexes that co-operates with Cdt1 to load MCM2-7Proteolyzed in yeasts or exported out of the nucleus in mammalian cells at the G1-S transition.Overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis,making it a critical regulator of DNA replication.MCM2-7:A family of six related proteins(MCM2-MCM7)which seem to function together in a large multi-subunit protein complex.The role of MCMs in DNA replication is still speculative,ranging from mediation of protein-DNA interactions to DNA helicase activity.,免疫学信息网http:/,The Xenopus Cdc6 Protein Is Essential for the Initiation of a Single Round of DNA Replication in Cell-Free Extracts Made from Xenopus egg extracts,Rxn Time:0-30 min30-60 min60-90 min,+,+,+,+,32P labeledDNAreplicationproduct,Replication initiation required ondouble-stranded DNA substrate,Only elongation required onsingle-stranded DNA substrate,免疫学信息网http:/,Orc is required to load CDC6;CDC6 is required to load MCM,免疫学信息网http:/,Immunostaining for CDC6 can pick outproliferating cells in Pap smears,免疫学信息网http:/,Pre-RC formation,ORC:A six subunit protein complex which has been implicated as being the eukaryotic DNA replication initiator protein.Subunits are named according to their size,with ORC1 being the largest and ORC6 being the smallest subunit.Yeast ORC specifically binds to replication origins in an ATP dependent manner and has been shown to possess ATPase activity.CDC6/Cdc18:An essential factor for the assembly of the pre-replicative complexes that co-operates with Cdt1 to load MCM2-7Proteolyzed in yeasts or exported out of the nucleus in mammalian cells at the G1-S transition.Overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis,making it a critical regulator of DNA replication.MCM2-7:A family of six related proteins(MCM2-MCM7)which seem to function together in a large multi-subunit protein complex.The role of MCMs in DNA replication is still speculative,ranging from mediation of protein-DNA interactions to DNA helicase activity.,免疫学信息网http:/,免疫学信息网http:/,Propidium iodide staining,No.of cells,Fluorescence analysis and cell sorting(FACS),DNA in cells stained with propidium iodide.Intensity of staining in each cell is proportional to amount of DNA in the cell.,免疫学信息网http:/,Chromatinimmunoprecipitation(ChIP)tests whether a given DNA sequence is bound by a protein in vivo,免疫学信息网http:/,免疫学信息网http:/,MCM homolog fromM.thermoautotrophicum(an archaebacterium)has helicase activity.What direction?,免疫学信息网http:/,Mt MCM formsdouble hexamers,免疫学信息网http:/,Pre-RC formation,ORC:A six subunit protein complex which has been implicated as being the eukaryotic DNA replication initiator protein.Subunits are named according to their size,with ORC1 being the largest and ORC6 being the smallest subunit.Yeast ORC specifically binds to replication origins in an ATP dependent manner and has been shown to possess ATPase activity.CDC6/Cdc18:An essential factor for the assembly of the pre-replicative complexes that co-operates with Cdt1 to load MCM2-7Proteolyzed in yeasts or exported out of the nucleus in mammalian cells at the G1-S transition.Overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis,making it a critical regulator of DNA replication.MCM2-7:A family of six related proteins(MCM2-MCM7)which seem to function together in a large multi-subunit protein complex.The role of MCMs in DNA replication is still speculative,ranging from mediation of protein-DNA interactions to DNA helicase activity.,免疫学信息网http:/,Mammalian cells can survive with 0.1x the normal ORC2 level:WT vs D/-cells,免疫学信息网http:/,oriP,EBNA1,EBNA1 protein,Replication from an origin in Epstein-Barr virus:a plasmid expressing EBNA1 and containing oriP willreplicate and be maintained as an episome inmammalian cells,Hygromycin-Resistancegene,免疫学信息网http:/,Establishment of drug-resistant coloniesafter transfection of EBV-based plasmid,