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免疫学信息网 http://www.immuneweb.com
Formation of pre-Replicative complex
免疫学信息网 http://www.immuneweb.com
G2M
ORC
MCM 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 http://www.immuneweb.com
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 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
A/T DUEOREAuxiliaryelements
Auxiliaryelements
Origin recognitionproteins
TranscriptionFactors
TranscriptionFactors
Leading str
Leading str
Lagging str
Lagging str
Core origin
Origins are multi-partite
免疫学信息网 http://www.immuneweb.com
The Structure of ARS1(S. cerevisiae)
B1 ACSB2B3
ABF1
ORC
“Post-RC” protection
“Pre-RC” protection
13
456
4 1
22
免疫学信息网 http://www.immuneweb.com
免疫学信息网 http://www.immuneweb.com
ORC uses different strategies for binding DNAin different species
免疫学信息网 http://www.immuneweb.com
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 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
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
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Orc is required to load CDC6;CDC6 is required to load MCM
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Immunostaining for CDC6 can pick outproliferating cells in Pap smears
免疫学信息网 http://www.immuneweb.com
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 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
免疫学信息网 http://www.immuneweb.com
Propidium iodide staining
No
. o
f ce
lls
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.
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Cross-link protein-DNA I.P. with anti MCM antibody
Reverse cross-link Purify DNA in I.P.
PCR to detect if your sequence was I.P.ed
MCM
anti-MCM Ab
Chromatinimmunoprecipitation
(ChIP) tests whether a given DNA sequence is bound by a protein
in vivo
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免疫学信息网 http://www.immuneweb.com
MCM homolog fromM. thermoautotrophicum(an archaebacterium)has helicase activity.
What direction?
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Mt MCM formsdouble hexamers
免疫学信息网 http://www.immuneweb.com
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 MCMís in DNA replication is still speculative, ranging from mediation of protein-DNA interactions to DNA helicase activity.
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Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs cells
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oriP
EB
NA
1
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 inmammalian cells
Hygromycin-Resistance
gene
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Establishment of drug-resistant coloniesafter transfection of EBV-based plasmid