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Formation of pre-Replicative complex

Formation of pre-Replicative complex. CDC6+ Cdt1. ORC. Replication Machinery. MCM. MCM. ORC. Replication complexes during the cell cycle. CDC6 + Cdt1. late G1. S. early G1. “Pre-Replicative Complex”. G2. M. Eukaryotic initiation complex.

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Formation of pre-Replicative complex

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  1. Formation of pre-Replicative complex 免疫学信息网http://www.immuneweb.com

  2. CDC6+ Cdt1 ORC Replication Machinery MCM MCM ORC Replication complexes during the cell cycle CDC6 + Cdt1 late G1 S early G1 “Pre-Replicative Complex” G2 M 免疫学信息网http://www.immuneweb.com

  3. 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 MCM2-7 Proteolyzed 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 MCMís in DNA replication is still speculative, ranging from mediation of protein-DNA interactions to DNA helicase activity. 免疫学信息网http://www.immuneweb.com

  4. Auxiliary elements A/T ORE DUE Auxiliary elements Transcription Factors Transcription Factors Origin recognition proteins Origins are multi-partite Core origin Leading str Lagging str Lagging str Leading str 免疫学信息网http://www.immuneweb.com

  5. 3 1 2 2 6 ABF1 4 5 4 1 “Post-RC” protection “Pre-RC” protection The Structure of ARS1(S. cerevisiae) ORC ACS B 3 B 1 B 2 免疫学信息网http://www.immuneweb.com

  6. 免疫学信息网http://www.immuneweb.com

  7. ORC uses different strategies for binding DNA in different species 免疫学信息网http://www.immuneweb.com

  8. 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 MCM2-7 Proteolyzed 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 MCMís in DNA replication is still speculative, ranging from mediation of protein-DNA interactions to DNA helicase activity. 免疫学信息网http://www.immuneweb.com

  9. 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 Only elongation required on single-stranded DNA substrate Replication initiation required on double-stranded DNA substrate 32P labeled DNA replication product Rxn Time: 0-30 min 30-60 min 60-90 min + + + + + + + + + + + + 免疫学信息网http://www.immuneweb.com

  10. Orc is required to load CDC6; CDC6 is required to load MCM 免疫学信息网http://www.immuneweb.com

  11. Immunostaining for CDC6 can pick out proliferating cells in Pap smears 免疫学信息网http://www.immuneweb.com

  12. 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 MCM2-7 Proteolyzed 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 MCMís in DNA replication is still speculative, ranging from mediation of protein-DNA interactions to DNA helicase activity. 免疫学信息网http://www.immuneweb.com

  13. 免疫学信息网http://www.immuneweb.com

  14. 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. No. of cells Propidium iodide staining 免疫学信息网http://www.immuneweb.com

  15. Chromatin immunoprecipitation (ChIP) tests whether a given DNA sequence is bound by a protein in vivo 免疫学信息网http://www.immuneweb.com

  16. 免疫学信息网http://www.immuneweb.com

  17. MCM homolog from M. thermoautotrophicum (an archaebacterium) has helicase activity. What direction? 免疫学信息网http://www.immuneweb.com

  18. Mt MCM forms double hexamers 免疫学信息网http://www.immuneweb.com

  19. 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 MCM2-7 Proteolyzed 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 MCMís in DNA replication is still speculative, ranging from mediation of protein-DNA interactions to DNA helicase activity. 免疫学信息网http://www.immuneweb.com

  20. Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs D/- cells 免疫学信息网http://www.immuneweb.com

  21. Replication from an origin in Epstein-Barr virus: a plasmid expressing EBNA1 and containing oriP will replicate and be maintained as an episome in mammalian cells EBNA1 protein oriP Hygromycin- Resistance gene EBNA1 免疫学信息网http://www.immuneweb.com

  22. Establishment of drug-resistant colonies after transfection of EBV-based plasmid 免疫学信息网http://www.immuneweb.com

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