This document summarizes DNA replication in eukaryotic cells. It describes that replication occurs through replicons to overcome the slower polymerases. Replication is initiated at specific sites called autonomous replicating sequences (ARS) where the origin recognition complex (ORC) binds. Elongation uses DNA polymerases α, δ, and ε and occurs semi-discontinuously with Okazaki fragments on the lagging strand. Termination involves removing RNA primers with RNase H and sealing fragments with DNA ligase. Multiple enzymes are involved in each phase including MCM helicase, primase, DNA ligase, and DNA polymerases.

1. DNA REPLICATION IN
EUKARYOTES
DR. ANU P. ABHIMANNUE
ASSISTANT PROFESSOR,
DEPARTMENT OF BIOTECHNOLOGY,
ST.MARY’S COLLEGE, THRISSUR.

2. REPLICATION
• Replication is the process by which DNA copies itself to
produce identical daughter molecules of DNA.
https://www.genome.gov/genetics-glossary/DNA-Replication
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3. EUKARYOTIC REPLICATION
• Eukaryotic genome is relatively complex and their
polymerases incorporate nucleotides at much slower rates.
• To overcome this, replication in eukaryotes occur in small
portions, termed replicons.
• In a human cell, there are about 10,000 to 100,000
replicons.
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4. REPLICONS IN EUKARYOTES
• Replicon was defined by
François Jacob; sydney
Brenner and François
Cuzin.
• It is the unit in which the
cell controls individual
acts of replication.
4
https://www.biology-pages.info/D/DNAReplication.html
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5. INITIATION
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6. ENZYMES INVOLVED
Sl no: Protein in initiation Role of protein
1 ORC Proteins (Multi protein
complex)
Recognition of origin of replication in
eukaryotes
2 Cdc6, Cdt1 Assist loading of helicase enzyme onto the DNA
3 Mcm (Mcm 2 – Mcm 7) DNA helicase enzyme that unwinds the double
stranded DNA into single strands.
Also called “licensing factors”
4 Replication Protein A (RPA) Binds to the exposed single stranded DNA
molecule and prevents its rewinding.
5 Topoisomerase I/II Relieves positive super coils located ahead of
the replication fork.
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7. AUTONOMOUS REPLICATING
SEQUENCES
• Specific site on DNA that functions like origin of replication
• Autonomous Replicating Sequences (ARS) consists of a
conserved sequence of 11 base pairs.
• A protein complex called Origin Recognition Complex (ORC)
binds DNA at ARS in a specific manner
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8. 1
• ORC specifically binds at ARS
2 • Cdc6 and Cdt1 – Binds ORC forming pre replication complex
3
• Mcm (“licensing factors”) bind at ORC and unwind DNA
4
• With the help of Cdk, DNA replication is initiated and
replication fork is formed.
5
• Single strand binding protein, Replication Protein A (RPA)
prevents rewinding of DNA
8
MECHANISM
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10. ELONGATION
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11. PROTEINS
Sl no Protein in
elongation
Role of protein
1 Primase Synthesizes RNA primers
2 RFC Subunits of DNA polymerase holoenzyme that load the clamp
onto the DNA
3 Pol δ/ε Primary replicating enzymes; synthesize entire leading strand
and Okazaki fragments; have proofreading capability
4 PCNA Ring-shaped subunit of DNA polymerase holoenzyme that
clamps polymerase to DNA
5 pol α Synthesizes short DNA oligonucleotides as part of RNA–DNA
primer
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12. DNA POLYMERASES
• Eukaryotic cells contain five DNA polymerases
• DNA polymerase α
• DNA polymerase β
• DNA polymerase γ
• DNA polymerase δ
• DNA polymerase ε
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13. DNA POLYMERASES - FUNCTIONS
• Polymerase γ is in mitochondria and replicates mitochondrial DNA.
• Polymerase β is required for DNA repair.
• polymerases α, δ and ε have replicative functions.
• Polymerase α is associated with primase, and initiate synthesis of
Okazaki fragment by adding 20 deoxyribonucleotides to the primer.
• Polymerase δ and ε is the primary DNA-synthesizing enzyme in the
lagging strand and leading strand respectively.
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14. DNA POLYMERASE
• The structure of eukaryotic and prokaryotic Polymerase
enzyme is quite similar.
• And consists of
– Sliding clamp called Proliferating Cell Nuclear Antigen
(PCNA).
– clamp loader that loads PCNA onto DNA is called
Replication Factor C (RFC).
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15. MECHANISM
Primase synthesize 10 bp long primer (RNA) and then
DNA polymerase α complex with primase and extend the
primer by adding 20-30 nucleotides
Primase - DNA polymerase α disocciates from DNA.
Binding of RFC at elongated primer to assist assembly of
PCNA. DNA polymerase δ binds to PCNA and elongates
Okazaki to a length of 150 - 200 bp.
By this time, enzyme reaches the previous Okazaki
fragment.
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16. POLYMERIZATION
• Eukaryotic polymerases elongate DNA strands in the 5'→3'
direction by the addition of nucleotides to a 3 hydroxyl group, and
none of them is able to initiate the synthesis of a DNA chain
without a primer.
• Eukaryotic DNA polymerase is present as a dimer, suggesting that
the leading and lagging strands are synthesized in a coordinate
manner by a single replicative complex, or replisome
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17. SEMI DISCONTINUOUS
• Leading strand in 5'→3 ‘ is continuous whereas lagging strand
in the opposite direction is dis-continuous
• Okazaki fragments are formed in the lagging strand.
• Okazaki fragments are smaller than in bacteria, averaging
about 150 nucleotides in length.
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18. TERMINATION
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19. PROTEINS
Sl no Protein in
termination
Role of protein
1 RNase H Cut RNA primers
2 Flap endonuclease
1 (FEN1)
Removes RNA primers; functions similar to pol I of E. coli
3 DNA ligase Seals Okazaki fragments into continuous strand
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20. MECHANISM
• RNA primer
removed from
the daughter
DNA
RNase H
FEN1
• Gap in the DNA
is filled by new
fragment
Polymerase δ
• Nick in the
DNA is sealed
to continous
strand
DNA ligase
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21. REFERENCES
• Gerald Karp (2010). Cell and molecular biology:
concepts and experiments (6th ed.). John Wiley &
sons. ISBN-13 978-0-470-48337-4.
• https://bio.libretexts.org/Bookshelves/Genetics/
Book%3A_Working_with_Molecular_Genetics_(H
ardison)/Unit_II%3A_Replication%2C_Maintenan
ce_and_Alteration_of_the_Genetic_Material/6._
DNA_replication_II%3A_Start%2C_stop_and_con
trol/6.1%3A_The_Replicon
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