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Formation of pre-Replicative complex


Replication complexes during the cell cycle CDC6+ Cdt1

ORC Replication Machinery CDC6 + Cdt1

MCM

late G1

S

MCM

“Pre-Replicative Complex”

early G1 G2

M

ORC


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 prereplicative 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.


Origins are multi-partite

Transcription Factors Auxiliary elements

Origin recognition proteins

A/T

ORE Core origin

Leading str Lagging str Lagging str

Leading str

Transcription Factors

DUE

Auxiliary elements


The Structure of ARS1 (S. cerevisiae) ORC 3 6

ABF1 B3

B2

22

5

4 1

B1

ACS

1

“Post-RC” protection “Pre-RC” protection

4


ORC uses different strategies for binding DNA in different species


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 prereplicative 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 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

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Orc is required to load CDC6; CDC6 is required to load M CM


Immunostaining for CDC6 can pick out proliferating cells in Pap smears


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 prereplicative 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.


Fluorescence analysis and cell sorting (FACS)

No. of cells

DNA in cells stained with propidium iodide. Intensity of staining in each cell is proportional to amount of DNA in the cell.

Propidium iodide staining


MCM

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

Cross-link protein-DNA I.P. with anti MCM antibody

anti-MCM Ab

Reverse cross-link Purify DNA in I.P.

PCR to detect if your sequence was I.P.ed


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


Mt M CM forms double hexamers


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 prereplicative 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.


Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs ∆/− cells


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 EBNA 1

Hygromyci nResistance gene


Establishment of drug-resistant colonies after transfection of EBV-based plasmid


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