Replication in eukaryotes

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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