How to ensure initiation of replication

only once per cycle ?

Rahna.K.RathnanAssistant professor

Sahrdaya College of engineering and technology

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.

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

The methylated DNA

Methylation state of DNA may regulate replication

GATCGATCCTAGCTAG

MeMe

MeMe

replicationreplication

GATCGATCCTAGCTAG

MeMe

GATCGATCCTAGCTAG

MeMehemimethylated DNAhemimethylated DNA

methylated DNAmethylated DNA

methylated DNAmethylated DNA

Dam methylaseDam methylase

Active Active originorigin

Inactive Inactive originorigin

Active Active originorigin

Sequestration

The membrane bound inhibitor binds to hemimethylated DNA

Remethylated DNA, inhibitor releases

DnaA binds to oriC--initiate replication

Methylation state of DNA may regulate replication

GATCnnnnnnnnnnnGATCnnnnnnnnnnnCTAGnnnnnnnnnnnCTAGnnnnnnnnnnn

MeMe Membrane-boundMembrane-bound inhibitor- competes with DnaA for oriCinhibitor- competes with DnaA for oriC

hemimethylated DNAhemimethylated DNA

GATCnnnnnnnnnnnGATCnnnnnnnnnnnCTAGnnnnnnnnnnnCTAGnnnnnnnnnnn

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))

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

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 !!

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), synthesisonly in the G1 phase

Cdc6, allow Mcm bind to complex

Replication initiation--Cdc6-Mcm are displaced

Yeast ARS region

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.

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.

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.

Fidelity of DNA replication

• One mistake per billion nucleotides

• How is this incredible accuracy is achieved?

– Proof reading of DNA polymerase

– Mismatch repair

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

Proof reading