Eukaryotic DNA replication

• More complex than prokaryotes

• DNA in chromatin form
• Needs dissociation and reassociation of
histones
• Every time nucleosome has to dissembled
• Rate of synthesis – 50 nucleotides/sec
 Proteins involved in replication
• DNA Polymerases α,δ,ε,γ.
• Helicase
• Topoisomerase
• Primase
• Ligase
• SSB Proteins
• dNTPs, Mg+2, & ATP
 DNApolymeraseAlpha

• - Located in nucleus
• - Catalysis the initiation of replication on both
leading and lagging strand synthesis
• - Tetramer – 4 subunits POLA 1 (catalytic) POLA 1
• (regulatory) POLA3 ,4 (Primase)
• - larger subunit - 5´-3´ polymerization activity
• -Two smaller subunit – primase activity
• - one subunit – assist in other three
• subunits
• - RNA primer 5-15 nucleotides are subsequently
• extended by DNAPol α.
 DNApolymerase Delta

• - Located in nucleus
• - Catalyzes the synthesis of lagging strand
• - Having four subunits – POLD 1,2,3,4
• - larger subunits catalyzes 5´-3´ polymerization
• activity
• - Smaller subunits catalyzes 3´-5´ exonuclease
• activity (proof reading activity)
• - High processivity when interacting with PCNA
• (Proliferating cell nuclear antigen).
 PCNA

• - Molecular weight 25,000;

• PCNA is important for both DNAsynthesis and DNArepair
• Multimeric protein - Found in large amount in nuclei of
proliferating cells. - Act as “clamp” to keep DNA pol δ from
dissociating off the leading DNA strand.
• “Clamp” consist of 3 PCNA molecules each containing two
topologically identical domains that are tightly associated to form
closed ring. - PCNA helps hold DNA polymerase epsilon (Pol ε) to
DNA.
• DNA pol δ improves fidelity of replication by a factor of 102 due to
its proof reading action. It contributes in limiting the rates of overall
error to 10-9 to 10-12. - DNAPol δ is also associated with helicase
activity.
 DNA polymerase Epsilon - Є
• located in nucleus
• Having four subunits – POLE 1, (Catalytic) 2,3,4
• (subunits)
• associated with - 5´- 3´ polymerization activity
• 5’- 3’ exonuclease activity (to remove RNAprimer)
• 3’- 5’ exonuclease activity (to proof read)
• DNA pol Є catalyzes the repair mechanism and catalyzes the removal of
primer and filing the primer gap in Okazaki fragments.
• Replicating factor A/ Replicating protein A (RPA/RFA)
• RPA/ RFA are similar to single strand binding protein. They bind to SS
DNA and prevent the reannealing of parental DNA.
 Replication factor C (RFC)

• RFC also called as clamp loader or matchmaker.

• RFC assist in DNA pol δ to form clampbetween
DNA and PCNA.
• RFC also plays important role in setting up a
link between DNA pol δ and DNA pol α, so that
the
• leading strand synthesis and lagging strand
synthesis
• in eukaryotes can take place simultaneously.
 ARS (Autonomously Replicating Sequences

• In eukaryotes the DNA replication is initiated at

• specific site known as ARS
• (Autonomously Replication Sequences) or replicators.
• ARS (Origin of chromosome in eukaryotes) contains
• - A central core sequence which contains highly
• conserved 11 bp sequence (AT rich sequence)
• - Flanking sequences. ARS – is 100- 150 long (generally it
span about 150 bp)
• There are multiple origins in eukaryotes
• In yeast 400
• Human chromosome contains average 100 ARS
 ARS
• Consists of ORE and DUE

• ORE – 11 basepair region

• Binds to DNA pol, helicase, rcf forms ORC

• DUE – adjacent to ORE 80 bp region

• ORC - (DNA pol α, DNA pol δ, RFC, PCNA,

RFA, SSB and helicase)
Formation of pre RC