Regulation of DNA replication

1. How to ensure initiation of
replication
only once per cycle ?
Rahna.K.Rathnan
Assistant professor
Sahrdaya College of engineering and technology

2. Three levels of control:
1.ATP hydrolysis by beta-clamp
2. Sequestration by a protein called
SeqA, that binds
hemimethylated DNA in oriC
3. Titration of DnaA and the dnaA
locus lowers the level of free DnaA
Control of DNA replication at oriC is complex
1.
2.
3.

3. Methylated DNA in the origin, can be
distinguished from the replicated DNA
In OriC, 11 copies of GATC
Parental strand is methylated
Daughter strands with
hemimethylated DNA, can
not be used to initiate a
replication cycle
Delay remethylation in oriC---delay replication

4. The methylated DNA

5. Methylation state of DNA may regulate
replication
GATCGATC
CTAGCTAG
MeMe
MeMe
replicationreplication
GATCGATC
CTAGCTAG
MeMe
GATCGATC
CTAGCTAG
MeMe
hemimethylated DNAhemimethylated DNA
methylated DNAmethylated DNA
methylated DNAmethylated DNA
Dam methylaseDam methylase
ActiveActive
originorigin
InactiveInactive
originorigin
ActiveActive
originorigin

6. Sequestration
The membrane bound
inhibitor binds to
hemimethylated DNA
Remethylated DNA,
inhibitor releases
DnaA binds to oriC
--initiate replication

7. Methylation state of DNA may
regulate replication
GATCnnnnnnnnnnnGATCnnnnnnnnnnn
CTAGnnnnnnnnnnnCTAGnnnnnnnnnnn
MeMe
Membrane-boundMembrane-bound inhibitor- competes with DnaA for oriCinhibitor- competes with DnaA for oriC
hemimethylated DNAhemimethylated DNA
GATCnnnnnnnnnnnGATCnnnnnnnnnnn
CTAGnnnnnnnnnnnCTAGnnnnnnnnnnn
MeMe
MeMe
DnaA proteinDnaA protein
Dam methylase (delayed)Dam methylase (delayed)
Active originActive origin
Inactive originInactive origin
~13 min.vs~13 min.vs
<1.5 min<1.5 min (dnaA promoter repressed;(dnaA promoter repressed;
also has delayed methylationalso has delayed methylation))

8. How to control the multiple replicons is
activated only once time in a single cycle ?

9. By rate-limiting component
which function only once at
the origin--licensing factor
Prevent more than one cycle
of replication--by removing
the component
Two purposes
Makes the replication
initiation dependent on cell
division
HOW !!

10. The licensing factor in Yeast
ORC: origin recognition complex, bind to A and B1 in ARS
Cdc6, a highly unstable protein (half-life ＜ 5 min), synthesis
only in the G1 phase
Cdc6, allow Mcm bind to complex
Replication initiation--Cdc6-Mcm are displaced

11. Yeast ARS region

13. positive control of replication
• each origin of replication must be bound by:
– an Origin Recognition Complex of proteins (ORC).
– These remain on the DNA throughout the process.
• Accessory proteins called licensing factors.
• These accumulate in the nucleus during G1 of the cell cycle. They
include:
– Cdc-6 and Cdt-1, which bind to the ORC and are essential for
coating the DNA with MCM proteins.
– Only DNA coated with MCM proteins (there are 6 of them) can be
replicated.

14. positive control of replication
• In Once replication begins in S phase,
• Cdc-6 and Cdt-1 leave the ORCs (the latter by ubiquination and
destruction in proteasomes).
• The MCM proteins leave in front of the advancing replication fork.

15. Geminin: negative control of
replication
• G2 nuclei also contain at least one protein — called geminin — that
prevents assembly of MCM proteins on freshly-synthesized DNA (probably
by blocking the actions of Cdt1).
• As the cell completes mitosis, geminin is degraded so the DNA of the two
daughter cells will be able to respond to licensing factors and be able to
replicate their DNA at the next S phase.

16. Fidelity of DNA replication
• One mistake per billion nucleotides
• How is this incredible accuracy is achieved?
– Proof reading of DNA polymerase
– Mismatch repair

17. Proof reading of DNA polymerase
• DNA pol remove nucleotides from a DNA chain, one at a time.
exonuclease activity
• Exonuclease activity improves the accuracy of DNA polymerase by
allowing the polymerase to back up and remove the last nucleotide
inserted into the DNA chain, if it is a wrong nucleotide.
• The polymerizing activity then puts in the correct nucleotide and
continues to elongate the new DNA strand.
• The combination of these two activities of DNA polymerase make the
copying of DNA very accurate

18. Proof reading