ATP

1
ATP
AMP-PNP
2
Immunostaining for CDC6 can pick
out proliferating cells in Pap smears
3
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. MCM 2-7 is most likely the
replicative DNA helicase. 
4
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
5
MCM8
MCM9
6
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
7
Chromatin immunoprecipitation (ChIP) tests
whether a given DNA sequence is bound by a
protein in vivo
8
(No Transcript)
9
Role of Pre-RC in mammalian cells
ORC CDC6/Cdc18 Cdt1 MCM2-7
10
Formation of pre-Replicative complex
Annual Review of Biochemistry 2002. Bell and
Dutta
11
Some cancers have an Achilles Heel
Viral episomes that carry viral
oncogenes Amplicons of cellular oncogenes, or of
drug resistance genes
Cancer
Normal
12
Human ORC
D/- cells
Wild type cells
ORC4
Quintana, 1997, 1998 Pinto/Quintana, 1999 Thome
2000 Dhar, 2000, 2001
13
Mammalian cells can survive with 0.1x the normal
ORC2 level WT vs D/- cells
14
Cell proliferation of ORC2 /, /- and D/- cells
measured by MTT assay
15
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
16
Establishment of drug-resistant colonies after
transfection of EBV-based plasmid
17
DpnI-resistance assays episome replication in
mammalian cells
Replicated once in human cells Hemi-methylated Dpn
I resistant
Replicated twice in human cells Unmethylated DpnI
resistant
Bacterial plasmid Methylated DpnI susceptible
18
1)EBV-based plasmid replication is decreased in
D/- cells. 2) Rescued by plasmid expressing ORC2D

19
Formation of pre-Replicative complex
Annual Review of Biochemistry 2002. Bell and
Dutta
20
(No Transcript)
21
Transition to replication from pre-RC to
unwinding
GINS complex Sld5-Go Psf1-Ichi Psf2-Ni Psf3-San
And-1/ CTF4
22
Transition to Replication
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?
23
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-transmissi
on-fidelity. Discovered in mammalian cells as a
nuclear protein! But found to co-immunoprecipitat
e 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.
24
Problems for the cell (1) Alternation of S and
M (2) Completion of S before M and vice versa
Mitosis Chromosome segregation
S phase Chromosome duplication
25
S and M have to alternate if not, genomic
instability
How does the cell prevent re-replication in the
same cycle?
1) cdks prevent licensing or pre-RC formation 2)
Any other mechanisms?
26
Control of Replication through the cell 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.
27
Positive factor present in S phase cells
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
28
Positive factor (replication licensing factor)
absent in G2 cells, but present in G1 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 phosphorylation Mcm10
degraded during G2 phase
29
CDC6 in mammalian cells is exported out of the
nucleus In S phase due to phosphorylation by
cyclin/cdk
Proper
Subcellular
Localizati
on of HsCDC6 is Dependent on
Both Intact
Phosphorylation
Sites and a Functional Cy Motif
G1
HsCDC6
G1
S
Wild-type
Ser

Ala
S
Ser
Asp

30
GFP-MCM4 localization through the cell cycle in
yeast
Movie http//users.ox.ac.uk/kearsey/mcm4.html
31
Negative factor present in G2 cells, but absent
in G1 cells
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.
32
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.

Geminin
33
Formation of pre-Replicative complex/ Licensed
chromatin
Bell and Dutta, ARB 2002
34
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
35
Geminin and Cdt1 Protein Levels During the Cell
Cycle
Time (hours)
0
3
6
9
12
21
18
15
24
Geminin
Cdt1
Cyclin A
Orc2
36
Cdt1
Geminin
37
Overexpression of Cdt1 and Cdc6 produces cells
with gt 4n DNA
BrdU incorporation
38
Cdt1
Geminin
39
HCT116(P53-/-)
4.8
Control RNAi
0.8
47
53.6
Gem4 RNAi
40
(No Transcript)
41
S and M have to alternate if not, genomic
instability
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)
42
Solution to the alternation problem
Low cyclin/cdk activity is required at G1/S to
initiate DNA replication Low cyclin/cdk activity
in G1/S is inadequate to initiate mitosis High
cyclin/cdk activity in G2 is required for
mitosis High cyclin/cdk activity in G2 inhibits
DNA replication
Solution to the completion problem
For mitosis to be completed, cyclin-cdk has to be
inactivated. Thus S phase cannot occur before
mitosis is completed. Why doesnt mitosis occur
before S phase is completed? Checkpoint pathways
that are still being worked out.
43
Cell Cycle Checkpoints
G1
S
G2
Metaphase
Anaphase
CDK1
CDK2
APC
ATR?Chk1
p53 ? p21
ATM?Chk2
Chromosome condensation
DNA replication Repair of damage
Repair of DNA damage
G1 DNA damage checkpoint
DNA replication checkpoint
Kinetochore attachment
S DNA damage checkpoint
Spindle checkpoint
44
Loss of geminin leads to re-replication and
activation of Chk1 and Chk2
HCT116
cont
Gem4
Loading control
Geminin
Phospho Chk1
Chk1
Phospho Chk2
Chk2
45
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.
46
S and M have to alternate if not, genomic
instability

• elevated activity of cdks
• 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