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.
Definition - Rolling circle replication is a process of unidirectional nucleic acid replication.
* can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids.
* Eucaryotic also replicate.
* widely used in molecular biology & biomedical
nanotechnology, especially in the field of
biosensing (as a method of signal Amplification).
Steps:
Circular ds DNA will be “nicked”
3` end is elongated →Leading strand
5` end displaced → Lagging strand
made up of double stranded by OKAZAKI fragments.
4) Replication of both “ unnicked” and displaced ss DNA
5) Displaced DNA circulates and synthesis its own complementary strand.
Initation-- phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins(SSBPs)
Elongation-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling Circle Model.
end will be displaced and will grow out like a waving thread.
Termination-* At the point of termination, the linear DNA molecule is cleaved from the circle resulting in a double stranded circular DNA molecule and a single- stranded linear DNA molecule.
* The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.
Example- Conjugation of F+ and F- bacteria
Diagrammatic representation of Rolling circle
some Examples-Viral DNA
* Human herpes virus
* Human papilloma virus
* Geminivirus
Viral RNA
* pospiviridiae
* Avsunviridiae
Reference:- https://en. m. wikipedia.org
what- when- how.com
https//www.sciencedirect.com
www.slideshare.com
Genetics-notes.wikispace.com
you tube
Prescott 5th edition page.no: 236, 237
Brock biology of microorganism , page.no: 253,616
DNA replication is the process by which DNA makes a copy of itself during cell division.The separation of the two single strands of DNA creates a 'Y' shape called a replication 'fork'. The two separated strands will act as templates for making the new strands of DNA.
Definition - Rolling circle replication is a process of unidirectional nucleic acid replication.
* can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids.
* Eucaryotic also replicate.
* widely used in molecular biology & biomedical
nanotechnology, especially in the field of
biosensing (as a method of signal Amplification).
Steps:
Circular ds DNA will be “nicked”
3` end is elongated →Leading strand
5` end displaced → Lagging strand
made up of double stranded by OKAZAKI fragments.
4) Replication of both “ unnicked” and displaced ss DNA
5) Displaced DNA circulates and synthesis its own complementary strand.
Initation-- phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins(SSBPs)
Elongation-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling Circle Model.
end will be displaced and will grow out like a waving thread.
Termination-* At the point of termination, the linear DNA molecule is cleaved from the circle resulting in a double stranded circular DNA molecule and a single- stranded linear DNA molecule.
* The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.
Example- Conjugation of F+ and F- bacteria
Diagrammatic representation of Rolling circle
some Examples-Viral DNA
* Human herpes virus
* Human papilloma virus
* Geminivirus
Viral RNA
* pospiviridiae
* Avsunviridiae
Reference:- https://en. m. wikipedia.org
what- when- how.com
https//www.sciencedirect.com
www.slideshare.com
Genetics-notes.wikispace.com
you tube
Prescott 5th edition page.no: 236, 237
Brock biology of microorganism , page.no: 253,616
DNA replication is the process by which DNA makes a copy of itself during cell division.The separation of the two single strands of DNA creates a 'Y' shape called a replication 'fork'. The two separated strands will act as templates for making the new strands of DNA.
A reaction in which daughter DNAs are synthesized using the parental DNAs as the template.
Transferring the genetic information to the descendant generation with a high fidelity
Semi-conservative replication
Bidirectional replication
Semi-continuous replication
High fidelity
Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand.
The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork.
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
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This is a presentation by Dada Robert in a Your Skill Boost masterclass organised by the Excellence Foundation for South Sudan (EFSS) on Saturday, the 25th and Sunday, the 26th of May 2024.
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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
2ANU P A, ST. MARY'S COLLEGE, THRISSUR
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.
3ANU P A, ST. MARY'S COLLEGE, THRISSUR
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
ANU P A, ST. MARY'S COLLEGE, THRISSUR
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.
6ANU P A, ST. MARY'S COLLEGE, THRISSUR
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
ANU P A, ST. MARY'S COLLEGE, THRISSUR
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
11ANU P A, ST. MARY'S COLLEGE, THRISSUR
12. DNA POLYMERASES
• Eukaryotic cells contain five DNA polymerases
• DNA polymerase α
• DNA polymerase β
• DNA polymerase γ
• DNA polymerase δ
• DNA polymerase ε
12ANU P A, ST. MARY'S COLLEGE, THRISSUR
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.
13ANU P A, ST. MARY'S COLLEGE, THRISSUR
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).
14ANU P A, ST. MARY'S COLLEGE, THRISSUR
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.
15ANU P A, ST. MARY'S COLLEGE, THRISSUR
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
16ANU P A, ST. MARY'S COLLEGE, THRISSUR
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.
17ANU P A, ST. MARY'S COLLEGE, THRISSUR
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
19ANU P A, ST. MARY'S COLLEGE, THRISSUR
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
20ANU P A, ST. MARY'S COLLEGE, THRISSUR
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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Editor's Notes
The most highly compacted,
least acetylated regions of the chromosome are packaged
into heterochromatin (page 485), and they are the last
regions to be replicated.
