DNA replication in Eukaryotes
- DNA replication is a part of eukaryotic cell cycle, happen in S-phase.
- But Initiated in Late G1 phase.
- Though they have many origin but replicates once and only once per cell cycle
- The fundamental mechanism of
eukaryotic replication is same as
prokaryotic DNA Replication but
some variation also there.
Process that are similar Include
- Formation of replication fork
- Semi-conservative replication
- Movement of replication fork
bidirectional
- Primer synthesis
- Okazaki fragment synthesis in lagging
strand
- Primer removal
- Gap bridging between newly synthesized
DNA fragments.
Difference between prokaryotic and eukaryotic replication
• Overall process of eukaryotic replication is bit more complex. Important differences are
due to
- Larger size of eukaryotic DNA (107- 1010 bases) compared to prokaryotic DNA 106 bases
in E. coli
- Distinct package of eukaryotic DNA in the term of chromatin
- Slower rate of fork movement in eukaryotes
- For DNA to become available to DNA polymerase, nucleotide must dissemble. This step
slows the Rate of fork movement.
Replication rate:
Prokaryotes: An E. coli replication fork progresses at approximately 1000 bp / sec.
Eukaryotes: Replication rate ten times slower than prokaryotes 50 nucleotides / sec.
Eukaryotic DNA polymerase:
In eukaryotes there are five different polymerases and they differ in
- Intracellular compartmentation
- Kinetic property
- Response to inhibitor
Processivity Low Low High High High
Fidelity High Low High High High
The fidelity of a DNA polymerase refers to its ability to accurately replicate a template.
PCNA (Proliferating cell nuclear antige)
- Molecular weight 25,000;
- PCNA is important for both DNA synthesis and DNA repair
- 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.
- DNA Pol δ is also associated with helicase activity
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 match maker.
- RFC assist in DNA pol δ to form clamp between
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.
Histone Dissociation and Association
-Since DNA is present in packaged form as chromatin, DNA replication is sandwiched
between two additional steps in eukaryotes.
1. Carefully ordered and complete dissociation of the chromatin.
2. Re-association of DNA with the histone octomers to form nucleosome.
Dissociation of histone: methylation at the fifth position of cytosine residues by a DNA
methyl transferase appears to functioning by loosening up the chromatin structure. This
allows DNA access to proteins and enzymes needed for DNA replication.
Synthesis of histone: the synthesis of
new histone occurs simultaneously
with DNA replication.
SEQUENTIAL STEPS IN EUKARYOTIC DNA REPLICATION
- DNA replication is a very complicated process that involves several enzymes and other
proteins. It occurs in following stages
• Pre-initiation
• Initiation
• Elongation
• Termination
• Telomerase function
PRE-INITIATION
- Actually during pre-initiation stage,
replicator selection occurs.
- Replicator selection is the process of
identifying the sequences that will direct
the initiation of replication and occur in G1
phase (prior to S phase).
- It involves formation of pre-replicative
complexes (pre-RCs).
- The first step is the recognition of the
replicator by the eukaryotic initiator, ORC
(Origin recognition Complex).
- Once ORC is bound, it recruits two helicase
loading proteins (cell division cycle protein
- Cdc6 and Cdtl).
- Together, ORC and the loading proteins
recruit a helicase (Mem 2-7 complex).
- The pre-RCs that are formed during G1 are
only activated to initiate replication after
cells pass from the G1 to the S phase of the
cell cycle.
INITIATION
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
1. A central core sequence which contains highly conserved 11 bp sequence (AT rich
sequence).
2. Flanking sequences (consist of overlapping sequence that include varients of core
sequences)
• There are multiple origins in
eukaryotes.
• Eg: yeast contains 400 ARS.
• The multiple origins are spaced
30 -300 kb apart.
• The sequence between two
origins of replication is called
replicons.
Initiation complex
- Formation of Pre-RC complex.
- At the transition of the G1 /S
phase, S phase–specific cyclin-
dependent protein kinase (CDK)
and Cdc7/Dbf4 kinase (DDK)
transform the pre-RC into an
active replication fork.
- pre-RC is disassembled with the MCM10 Cdc45
MCM10
loss of Cdc6, creating the initiation GINS
complex.
- At onset of S-pahse, Mcm loads
Cdc45 onto chromatin.
- The loading of Cdc45 onto MCM10
MCM10
chromatin is critical for loading
other proteins, including DNA
polymerase α, DNA polymerase
ε, RPA) and PCNA onto chromatin.
- GINS are essential for the interaction of Mcm and Cdc45 at the origins of replication during
initiation and elongation.
- GINS (go, ichi, ni, san' which means '5, 1, 2, 3' in Japanese) is complex of 4 proteins Sld5 (Cdc105), Psf1
(Cdc101), Psf2 (Cdc102) and Psf3 (Cdc103).
- Cdc45, Mcm2-7 and GINS together form the CMG helicase, thus presence of Cdc45 and GINS are
required for robust helicase activity.
Elongation
- All but one of the ribonucleotides in RNA primer is removed by RNase H1.
- Then exonuclease activity of FEN 1/ RTH 1 complex removes the one remaining
nucleotide.
- The gap is filled by DNA pol Є by its 5’-3’ polymerase activity.
- DNA ligase joins the Okazaki fragment of the growing DNA strand.
TERMINATION
- When the replication forks meet each other,
then termination occurs.
- It will result in the formation of two duplex
DNA.
- Even though replication terminated, 5’ end of
telomeric part of the newly synthesized DNA
found to have shorter DNA strand than the
template parent strand.
- This shortage corrected by the action of
telomerase enzyme and then only the actual
replication completed.
TELOMERES
- Eukaryotic chromosomes are linear.
- The ends of chromosomes have specialized
structures known as ‘Telomeres’.
- Telomeres are – short (5-8 bp) - tandem repeated
and - GC rich nucleotide sequence.
- Telomeres form protective cap 7-12 kbp long in the
ends of chromosome.
- Telomeres are necessary for chromosome
maintenance and stability.
- They are responsible for maintaining chromosome
integrity by protecting against DNA degradation
and rearrangement.
Problem in the completion of replication of lagging strand:
- Linear genomes including those of several viruses as well as the chromosomes of
eukaryotic cells force a special problem completion of replication of the lagging strand.
- Excision of an RNA primer from the 5’ end of a linear molecule would leave a gap (primer
gap).
- This gap cannot be filled by DNA polymerase action, because of the absence of a primer
terminus to extend.
- If the DNA could not be replicated, the chromosome would shorten a bit with each
round of replication during cell cycle.
- This problem has been solved by Telomerase.
Telomerase:
- Telomeric DNA consists of simple tandemly
repeated sequences like those shown as below:
- Telomeric repeats sequence at 5’end
- Telomerase is ribonucleoprotein. It contains
an RNA component which has repeat of 9 to
30 nucleotides long.
- This RNA component serves as the template
for the synthesis of telomeric repeats at the
parental DNA ends.
- Telomerase is a RNA dependent DNA
polymerase with a RNA component.
- Telomerase uses the - 3’ end of parental
DNA strand as primer,
- RNA component of telomerase as
template,
- adds successive telomeric repeats to the
parental DNA strand at its 3’ end due to its
5’-3’ RNA dependent DNA polymerase
activity, then the telomerase is released.
- Finally the RNA primer, (of telomerase) is bound
near the lagging strand and it is extended by DNA
polymerase.
-Thus the lagging strand synthesis is completed.
