Transition to replication: from preRC to unwinding

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Transition to replication from pre-RC to
unwinding
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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 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 act
together with ORC and the 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?
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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
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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.
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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
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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 ?
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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

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GFP-MCM4 localization through the cell cycle in
yeast
Movie http//users.ox.ac.uk/kearsey/mcm4.html
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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.
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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 inhibits DNA
replication High cyclin/cdk activity in G2 is
required for mitosis
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.
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