The document summarizes regulation of DNA replication in eukaryotes. It explains that eukaryotic genomes are divided into replicons that are each activated once per cell cycle. This is achieved through licensing factors that load onto origins of replication in G1 phase, but are removed or inactivated during DNA replication, preventing re-replication. The key licensing factors are the origin recognition complex (ORC) and proteins Cdc6 and Cdt1, which load the MCM complex onto DNA.

1. DNA replication:
Regulation in
eukaryotes
Jahnvi Sharma- 22MBT013
Jinal Prajapati- 22MBT014

2. Table of content
2
1. How is regulation achieved?
2. Licensing factor controls eukaryotic re-
replication
3. Binding of licensing factors to ORC.
4. Learning outcomes
5. References

3. “
How is regulation in
Eukaryotic replication
achieved?
3

4. Eukaryotic genome is divided into multiple replicons
Origin in each replication is activated only
and only once in single division cycle
Achieved by
Providing some
rate-limiting
component that
functions only at
an origin
By presence of
repressor that
prevent
rereplication at
origin that have
been used

5. 5
How system determines whether any particular origin has
been replicated and what protein are involved?
Xenopus egg
Insight into the nature of protein components have been provided by using a
system in which substrate DNA undergoes only one cycle of replication
• It has all the components needed to replicate
DNA
• In first few hours after fertilization they
undertake 11 division cycle without new gene
expression
• They can replicate the DNA in a nucleus that is
injected into egg

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Sperm or interphase nucleus DNA is replicated only
once in presence of heavy precursor
If protein synthesis is blocked in the
egg, the membrane around the
injected material remain intact and
DNA cannot replicate again.
In the presence of protein synthesis,
nuclear membrane breaks down just like
in normal cell division and subsequent
replication cycle can occur. The same
result can be achieved by using nuclear
membrane permeabilizing agents
This suggests that nucleus contains a protein(s) needed for replication that is used up in some way, so even
though more protein is present in cytoplasm, it can only enter nucleus if the nuclear membrane breaks down.

7. “
Licensing factor
controls
Eukaryotic Re
Replication
7

8. 8
This regulatory system achieves two
purposes
By removing
necessary
component after
replication, it
prevents more than
one cycle of
replication from
occurring.
It provides
feedback loop
that makes the
initiation of
replication
dependent on
passing through
the cell cycle.

9. “
Binding of
licensing factors
to ORC
9

10. 10
Origin Recognition Complex (ORC)
It is protein complex
that is associated with
yeast origins
throughout the cell
cycle
In S. Cerevisiae, ORC is 400 KD
complex that binds to ARS sequence.
ARS consists of
• A consensus sequence
• 3 B element
ORC complex is of 6
protein
It binds to A and adjacent B1 element.
ORC2-5 binds strongly while ORC6 binds
weakly and has nuclear localizing signal.
ORC must be activated by cyclin/CDK kinase
during G1 to S transition.
ORC remains bound at the
origin through the entire cell
cycle. However, changes
occur in the pattern of
protection of DNA as a result
of binding of other protein to
the ORC origin complex

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Transcription factor
ABF1
Binds to B3
element of ARS
This affects chromatin
structure and assists
initiation
But it is the event that
occur at A and B1 element
which actually cause
initiation
• Most origins are localized in regions between genes, which
suggests that it may be important for the local chromatin structure
to be in a nontranscribed condition.

12. 12
Origin (ARS)
Cdc6 Cdt1
In yeast, it is highly unstable protein
with half life of <5 minutes. It is
synthesized during G1 and binds to
ORC between the exit from mitosis
and late G1. It’s rapid degradation
means that no protein is available
later in cycle
In mammalian cells, it is controlled
differently. It is phosphorylated during
S phase and as a result, it is degraded
by the ubiquitination pathway.
Geminin
Cdt1 is initially stabilized by
the protein Geminin, which
prevent its degradation and
subsequent Geminin binding
prevents its reuse
• This feature make Cdc6 and Cdt1 a key licensing factors.
• This two proteins also provide connection between ORC and other complex of protein involved in
initiation of replication.
• Cdc6 has an ATPase activity that is required for it to support initiation.

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Cdc6 Cdt1
MCM
The presence of Cdc6 and Cdt1 is
necessary for initiation as it allows
MCM protein to bind to complex in G1
stage in inactivate state.
PRE-REPLICATION
COMPLEX – origin
enters S phase.
• In yeast, the replication helicase MCM 2-7 complex enters the nucleus as an inactive
double header during mitosis.
• MCM 2-7 protein forms a 6 membered ring complex around DNA.
• MCM 2,3,5 are regulatory, while MCM 4,6,7 have helicase activity.

14. 14
Cdt1
Cdc6
Postreplication complex –
when initiation occurs
Cdc6
Cdt1
• It is rapidly degraded during S phase and as a result it is not available to
support reloading of MCM protein. Thus, origin cannot be used for 2nd
cycle of initiation during S phase.
• If it is made available to bind to the origin during G2, MCM protein do
not bind until the following G1, which suggests that there is a secondary
mechanism to ensure that they associate with origins only at the right
time. This could be another part of licensing control.
• In mammalian cells, Cdt1 is targeted for degradation by
action of protein complex that is recruited to origin of
replication by PCNA, the eukaryotic counterpart of bacterial
β-clamp.

15. 15
• In S. cerevisiae, control at level of nuclear entry is not seen and this could be difference
between yeast and animal cells. Some of the ORC protein have similarities to replication
proteins that load DNA polymerase onto DNA. It is possible that ORC uses hydrolysis of
ATP to load the MCM ring onto DNA.
• In Xenopus extract, replication can be initiated if ORC is removed after if has loaded
Cdc6 and MCM proteins. This shows major role of ORC is to identify the origin to the
Cdc6 and MCM protein that control initiation and licensing.
• As the transition from G1 to S phase begins,
CDK/Cyclin recruits Cdc45 and the GINS
complex to MCM helicase, which then
becomes known as the CMG complex for
activation.
• This marks the transition from initiation to
DNA replication, that is, the elongation phase
of replication that entails the two different
modes of synthesis on leading and lagging
strand.

16. “
Learning
outcomes
16

17. Learning outcomes
17
• Licensing factor is necessary for initiation of replication at each origin.
• Licensing factor is present in nucleus prior to replication, but is
removed, inactivated or destroyed by replication.
• Initiation of another replication cycle becomes possible only after
licensing factor reenters the nucleus after mitosis.
• ORC is a protein complex that is associated with yeast origins
throughout the cell cycle.
• Cdc6 Protein is an unstable protein that is synthesized only in G1.
• Cdc6 binds to ORC and allows MCM loading on origins.
• When replication is initiated, Cdc6 and Cdt1 are displaced. The
degradation of Cdc6 prevents reinitiation.

18. “
References
18

19. References
19
• Lewin’s GENES XI by Jocelyn E. Krebs, Elliott S. Goldstein and Stephen T. Kilpatrick
(Section 12.10 to 12.12)
• Cristofori-Armstrong, B., Soh, M., Talwar, S. et al. Xenopus borealis as an alternative
source of oocytes for biophysical and pharmacological studies of neuronal ion channels. Sci
Rep 5, 14763 (2015). https://doi.org/10.1038/srep14763
• Nicola J. Gardner, Peter J. Gillespie, Jamie T. Carrington, Emma J. Shanks, Stuart P.
McElroy, Emma J. Haagensen, Julie A. Frearson, Andrew Woodland, J. Julian Blow, The
High-Affinity Interaction between ORC and DNA that Is Required for Replication Licensing
Is Inhibited by 2-Arylquinolin-4-Amines, Cell Chemical Biology, Volume 24, Issue 8, 2017,
Pages 981-992.e4, ISSN 2451-9456, https://doi.org/10.1016/j.chembiol.2017.06.019.
(https://www.sciencedirect.com/science/article/pii/S2451945617302337)
• Elisa Ventura, Antonio Giordano, Cell Cycle, Reference Module in Life Sciences, Elsevier,
2019, , ISBN 9780128096338, https://doi.org/10.1016/B978-0-12-809633-8.90189-4.
(https://www.sciencedirect.com/science/article/pii/B9780128096338901894)

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Thank you!