ATP

AMP-PNP

Immunostaining for CDC6 can pick outproliferating cells in Pap smears

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.MCM 2-7 is most likely the replicative DNA helicase. 

Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes)

Yeast containing Leu2 on a plasmid

Plate yeast on Leucine minus plates to estimate rate of plasmid loss

Grow under non-selective conditions

MCM8

MCM9

Form hexamer

Associates with MCM2-7

Newbies of unknown function

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

Role of Pre-RC in mammalian cells

ORC :CDC6/Cdc18 : Cdt1:MCM2-7 :

Formation of pre-Replicative complex

Annual Review of Biochemistry2002. Bell and Dutta

Some cancers have an Achilles Heel

Viral episomes that carry viral oncogenesAmplicons of cellular oncogenes, or of drug resistance genes

Normal Cancer

Human ORC

Quintana, 1997, 1998Pinto/Quintana, 1999Thome 2000Dhar, 2000, 2001

ORC4

Wild type cells /- cells

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

Cell proliferation of ORC2 and cells measured by MTT assay

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

m

m

mm

m

m

DpnI

DpnI

DpnI

DpnI

m

m

m

DpnI

DpnI

X

X

X

DpnI

DpnI

DpnIX

X

X

Bacterial plasmidMethylatedDpnI susceptible

Replicated once inhuman cellsHemi-methylatedDpnI resistant

Replicated twice inhuman cellsUnmethylatedDpnI resistant

DpnI-resistance assays episome replication in mammalian cells

1)EBV-based plasmid replication is decreased in /- cells. 2) Rescued by plasmid expressing ORC2

+ +

Formation of pre-Replicative complex

Annual Review of Biochemistry2002. Bell and Dutta

Transition to replication: from pre-RC to unwinding

GINS complexSld5-GoPsf1-IchiPsf2-NiPsf3-San

And-1/CTF4

MCM10 : essential function in the initiation of DNA replication in yeast. Required in Xenopus after the loading of Mcm2-7, before the loading of CDC45. In yeast, depletion of MCM10 causes the DNA polymerase alpha, catalytic subunit (p180), to be degraded. In yeast associates with DNA pol alpha p180. In mammalian cells associates with DNA pol alpha p180 and loads it on chromatin through the recruitment of AND1/CTF4

CDC7/DBF4 : A DBF4 dependent kinase (DDK) which, together with Dbf4, is required for the initiation of DNA replication. Possible substrates of this kinase are the MCMs. How do we know this?

Cdk/Cyclin: A protein kinase essential for the initiation of DNA replication. Substrates that are activated by Cdk for replication initiation are unclear. MCM, CDC6 and some ORC subunits are phosphorylated, but this phosphorylation inactivates the proteins and prevents re-replication.

CDC45 : it seems to activate MCMs to initiate DNA replication.Has a role in the loading of DNA primase/polymerase complex.It and the MCM proteins appear to move along with the replication fork. How do we know this?

Transition to Replication

GINS : essential function in the initiation of DNA replication in yeast. Required in Xenopus after the loading of Mcm2-7, before the loading of CDC45. Composed of 4 subunits: SLD5, PSF1, PSF2 and PSF3 (the Go-Ichi-Ni-San or GINS complex). Appears to form a ring on EM

AND1/CTF4: Discovered in yeast because mutations lead to defects in chromosome-transmission-fidelity. Discovered in mammalian cells as a nuclear protein! But found to co-immunoprecipitate with Mcm10 and with p180 subunit of DNA pol alpha. Required for the stability of p180 and also to load p180 on chromatin at origins of replication.

MCM8: Related to MCM2-7 in sequence and has a helicase domain. No yeast homolog. Depletion in Xenopus replication reaction decreases DNA replication by 50% without affecting pre-RC formation. Controversy in human cells, where RNAi of MCM8 has been reported to decrease pre-RC formation.

MCM9: Similar to MCM8. Depletion in Xenopus replication reaction decreases pre-RC formation. RNAi in human cells affects cell proliferation.

Transition to Replication

S phase:Chromosomeduplication

Mitosis:Chromosomesegregation

Problems for the cell: (1) Alternation of S and M(2) Completion of S before M and vice versa

S and M have to alternate: if not, genomic instability

S

M

G1 G2

1) cdks prevent licensing or pre-RC formation

2) Any other mechanisms?

How does the cell prevent re-replication in the same cycle?

With their multiple origins, how does the eukaryotic cell know which origins have been already replicated and which still await replication?

Two observations:When a cell in G1of the cell cycle is fused with a cell in S phase, the DNA of the G1 nucleus begins replicating early. Thus S phase cells have a positive factor for DNA replication that G1 cells lack.

When a cell in G2 of the cell cycle is fused with a cell in S phase, the DNA of the G2 nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again. Thus cells in G2 have a negative factor that disappears in mitosis and/or lack a positive replication licensing factor until they pass through mitosis.

Control of Replication through the cell cycle

Cyclin/cdk protein kinase (i) yeast with temperature sensitive mutation in CDC28 (a cdk) arrest in G1(ii) addition of a cdk inhibitor (e.g. p21) to Xenopus egg extracts in G1 inhibits replication

DBF4/CDC7 protein kinase(i) ts mutation of CDC7 in yeast blocks cell cycle in G1 (by-passed by a mutation in MCM5)(ii) antibody mediated depletion in Xenopus egg extracts

Positive factor present in S phase cells

CDC6(i) degraded in yeasts after G1/S(ii) displaced out of the nucleus after G1/S in mammalian cells

MCM2-7(i) displaced out of the nucleus after G1/S in yeast(ii) loses tight association with chromatin after G1/S in mammalian cells

CDC45(i) loses tight association with chromatin after G1/S in mammalian cells

Cdt1(i) degraded in mammalian cells during S phase.(ii) inactivated by interaction with a protein called geminin.

ORC: may be inactivated by phosphorylationMcm10: degraded during G2 phase

Positive factor (replication licensing factor) absent in G2 cells, but present in G1 cells

HsCDC6 G1 S

H2N COOHCy

S S

Wild-type

H2N COOHCy

S S

Cy

H2N COOHCy

D D

H2N COOHCy

A A

Ser Ala

Ser Asp

Proper Subcellular Localizati on of HsCDC6 is Dependent onBoth Intact Phosphorylation Sites and a Functional Cy Motif

CDC6 in mammalian cells is exported out of the nucleusIn S phase due to phosphorylation by cyclin/cdk

G1

S

GFP-MCM4 localization through the cell cycle in yeast

Movie: http://users.ox.ac.uk/~kearsey/mcm4.html

Highly active cyclin/cdk protein kinase(i) inactivated in mitosis due to degradation of cyclins. (ii) artificial inactivation in G2 yeast with ts mutations in the cdk or by induction of a cdk inhibitor results in re-replication of DNA without mitosis.(iii) the nuclear export (in mammalian cells) or proteolysis (in yeast) of CDC6 is due to phosphorylation by cyclin/cdk. Therefore the negative factor present in G2 cells may also be responsible for the absence of a positive replication licensing factor in G2 nuclei!

Geminin(i) elevated in G2 cells and degraded in mitosis(ii) addition of a stable form of the protein to Xenopus egg extract inhibits the loading of MCM2-7 on chromatin and prevents DNA replication(iii) interacts with Cdt1.

Negative factor present in G2 cells, but absent in G1 cells

Discovery of Geminin

Identified in Xenopus as a protein degraded by the Anaphase Promoting Complex during the Metaphase to Anaphase Transition

Inhibits the initiation of DNA replication by blocking the loading of MCM proteins onto replication origins.

ORCCDC6

ORC

CDC6

MCM

Geminin

Formation of pre-Replicative complex/ Licensed chromatin

Bell and Dutta, ARB 2002

Geminin-Cdt1

•Geminin interacts with and inhibits Cdt1

•High levels of geminin in the latter half of the cell-cycle might prevent re-replication by inhibiting Cdt1

•Is geminin an inhibitor of the cell-cycle? A tumor suppressor? Does it really prevent re-replication?

Wohlschlegel, Science, 2000

Orc2

Cyclin A

Cdt1

Geminin

Time (hours)0 3 6 9 12 211815 24

Geminin and Cdt1 Protein Levels During the Cell Cycle

Geminin

Cdt1

Overexpression of Cdt1 and Cdc6 produces cells with > 4n DNA

Ce

ll n

um

be

r

DNA content DNA content

Brd

U

inc

orp

ora

tio

n

Geminin

Cdt1

HCT116(P53-/-)

Control RNAi

Gem4 RNAi

4.8%

53.6%

0.8%

47%

S and M have to alternate: if not, genomic instability

S

M

G1 G2

elevated activity of cdks

elevated level of geminin

assembly of pre-RC can only occur in a window in G1 (Cdc6 exported, Cdt1 degraded, Mcm2-7 phosphorylated in S)

Low cyclin/cdk activity is required at G1/S to initiate DNA replicationLow cyclin/cdk activity in G1/S is inadequate to initiate mitosis

High cyclin/cdk activity in G2 is required for mitosisHigh cyclin/cdk activity in G2 inhibits DNA replication

Solution to the alternation problem

Solution to the completion problem

For mitosis to be completed, cyclin-cdk has to be inactivated. Thus S phase cannotoccur before mitosis is completed.

Why doesn’t mitosis occur before S phase is completed? Checkpoint pathways thatare still being worked out.

CDK1

Chromosomecondensation

Sister chromatidseparation

CDK2

DNA replication;Repair of damage

APC

DNA replicationcheckpoint

Spindlecheckpoint

S G2 Metaphase Anaphase

Kinetochoreattachment

Repair of DNA damage

G1

G1 DNA damagecheckpoint

Cell Cycle Checkpoints

p53 p21

S DNA damagecheckpoint

ATRChk1ATMChk2

HCT116cont Gem4

PhosphoChk1

PhosphoChk2

Chk1

Chk2

Loadingcontrol

Geminin

Loss of geminin leads to re-replication and activation of Chk1 and Chk2

Depletion of geminin activates G2/M checkpoint,resulting in sequestration of Cdc25C outside the nucleus (red on right panel: cytoplasmic Cdc25C).

Rereplication by depletion of geminin activates the G2/M checkpoint.

S and M have to alternate: if not, genomic instability

S

M

G1 G2

1) elevated activity of cdks

2) elevated level of geminin

3) assembly of pre-RC can only occur in a window in G1 (Cdc6 exported, Cdt1 degraded, Mcm2-7 phosphorylated in S)

4) If despite this re-replication occurs: checkpoint pathways stop the cell-cycle