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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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slide1

Formation of pre-Replicative complex

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replication complexes during the cell cycle

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

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slide3

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.

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slide4

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

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the structure of ars1 s cerevisiae

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

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slide7

ORC uses different strategies for binding DNA

in different species

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slide8

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.

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slide9

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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slide10

Orc is required to load CDC6;

CDC6 is required to load MCM

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slide11

Immunostaining for CDC6 can pick out

proliferating cells in Pap smears

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slide12

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.

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slide14

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

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slide15

Chromatin

immunoprecipitation

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

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slide17

MCM homolog from

M. thermoautotrophicum

(an archaebacterium)

has helicase activity.

What direction?

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slide18

Mt MCM forms

double hexamers

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slide19

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.

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slide20

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

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slide21

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

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slide22

Establishment of drug-resistant colonies

after transfection of EBV-based plasmid

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