免疫学信息网 Formation of pre-Replicative complex.

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Formation of pre-Replicative complex


G2M

ORC

MCM MCM

CDC6 +Cdt1

Replication Machinery

“Pre-Replicative Complex”

Replication complexes during the cell cycle

ORC

CDC6+Cdt1

Slate G1

early G1


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.


A/T DUEOREAuxiliaryelements

Auxiliaryelements

Origin recognitionproteins

TranscriptionFactors

TranscriptionFactors

Leading str

Leading str

Lagging str

Lagging str

Core origin

Origins are multi-partite


The Structure of ARS1(S. cerevisiae)

B1 ACSB2B3

ABF1

ORC

“Post-RC” protection

“Pre-RC” protection

13

456

4 1

22


ORC uses different strategies for binding DNAin different species


Pre-RC formation





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


Orc is required to load CDC6;CDC6 is required to load MCM


Immunostaining for CDC6 can pick outproliferating cells in Pap smears


Pre-RC formation





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.


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


MCM homolog fromM. thermoautotrophicum(an archaebacterium)has helicase activity.

What direction?


Mt MCM formsdouble hexamers


Pre-RC formation





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


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


Establishment of drug-resistant coloniesafter transfection of EBV-based plasmid

免疫学信息网 Formation of pre-Replicative complex.

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