免疫学信息网 Formation of pre-Replicative complex

免疫学信息网 G2 M ORC MCM CDC6 + Cdt1 Replication Machinery “Pre-Replicative Complex” Replication complexes during the cell cycle ORC CDC6+ Cdt1 Slate G1 early G1

免疫学信息网 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 MCM í s in DNA replication is still speculative, ranging from mediation of protein-DNA interactions to DNA helicase activity.

免疫学信息网 A/TDUEORE Auxiliary elements Auxiliary elements Origin recognition proteins Transcription Factors Transcription Factors Leading str Lagging str Core origin Origins are multi-partite

免疫学信息网 The Structure of ARS1 (S. cerevisiae) B1 ACS B2 B3 ABF1 ORC “Post-RC” protection “Pre-RC” protection

免疫学信息网

ORC uses different strategies for binding DNA in different species

免疫学信息网 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 MCM í s in DNA replication is still speculative, ranging from mediation of protein-DNA interactions to DNA helicase activity.

免疫学信息网 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 min min min + 32P labeled DNA replication product Replication initiation required on double-stranded DNA substrate Only elongation required on single-stranded DNA substrate

免疫学信息网 Orc is required to load CDC6; CDC6 is required to load MCM

免疫学信息网 Immunostaining for CDC6 can pick out proliferating cells in Pap smears

免疫学信息网 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 MCM í s in DNA replication is still speculative, ranging from mediation of protein-DNA interactions to DNA helicase activity.

免疫学信息网

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 Chromatin immunoprecipitation (ChIP) tests whether a given DNA sequence is bound by a protein in vivo

免疫学信息网

MCM homolog from M. thermoautotrophicum (an archaebacterium) has helicase activity. What direction?

免疫学信息网 Mt MCM forms double hexamers

免疫学信息网 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 MCM í s in DNA replication is still speculative, ranging from mediation of protein-DNA interactions to DNA helicase activity.

免疫学信息网 Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs  cells

免疫学信息网 oriP EBNA1 EBNA1 protein 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 Hygromycin- Resistance gene

免疫学信息网 Establishment of drug-resistant colonies after transfection of EBV-based plasmid