This document discusses DNA replication. It begins by defining replication and describing the main types (conservative, dispersive, semiconservative). It then discusses the key proteins and enzymes involved like DNA polymerase, helicase, topoisomerase, primase, and ligase. The main stages of replication - initiation, elongation, and termination - are outlined. Initiation involves unwinding the DNA and forming a replication fork. Elongation describes continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand in short Okazaki fragments.

1. replication1.swf

2. DNA Replication

3. OBJECTIVES
1. WHAT IS REPLICATION ?
2. WHAT ARE THE DIFFERENT TYPES OF REPLICATION?
3. What are different modes of Replication?
4. What type of replication is eukaryotic?
5. What are the proteins and enzymes involved in replication?
6. What are their functions?

4. 8. What are the sequence of events that occurs in
replication?
9. What is lagging strand and leading strand?
10, What are okazaki fragments ?

5. 06_04_replic.rounds.jpg

6. DNA Replication
Replication = DNA copies itself
exactly
(Occurs within the nucleus)
 Any mistake in copying = mutation
 DNA mutation = chromosomal
mutation

7. • Conservative replication model
• Dispersive replication model
• Semiconservative replication
Proposed DNA Replication Models

9. •Theta replication: circular DNA, E. coli; single
origin of replication forming a replication fork,
usually a bidirectional replication
Modes of Replication

10. Rolling-circle replication: virus, F factor of E. coli;
single origin of replication

11. Eukaryotic cells; thousands of origins; a typical replicon:
200,000 ~ 300,000 bp in length.
Linear Eukaryotic Replication

12. BASIC FACTS OF REPLICATION

13. The strand acting as template is read from 3’
to 5’ direction.
Both strands can be replicated simultaneously.
One strand is synthesized continuously and the
other strand is synthesized discontinuously.
Replicons: units of replication

15. Replication begins at an origin and usually proceeds
Bidirectionally.
Unwind at one point and use that as the origin
of replication.
Region is AT rich to allow easy separation.
Eukaryotes have multiple replication origins
(Humans = 10,000).

17. Replication fork:
Where the parental DNA is being unwound and
separated strands quickly replicates.

18. DNA synthesis proceeds in a 5’ – 3’ direction and it
Is semidiscontinous.

19. Continuous strand: Leading strand proceeds in the
Same direction as the replication fork.
Discontinuous strand : Okasaki fragment or the
Lagging strand. Opposite to the direction of
The replication fork.

21. Enzymes and proteins required for DNA replication
20 or more different enzymes and proteins are
Necessary for DNA replication.
These are called as DNA replicase system or
Replisome.

23. Helicases : Moves along the DNA and separate
Strands using chemical energy from ATP.
Topoisomerase : Relieves the topological stress
Induced by strand separation.
DNA binding proteins: The separated strands are
Stabilized by DNA binding proteins.
Primases: The primers are generally short segments
Of RNA laid down by enzymes called primases.

24. DNA ligases:
The RNA primers must be removed and replaced by DNA.
After removal of the RNA segments and filling in the
Gap with DNA there remains points in DNA backbone
Where a phosphodiester bond is broken.
This is sealed by ligases.

25. Basic Requirements for replication
Substrates
Four deoxyribonucleotides
dATP
dGTP
dCTP
dTTP

26. TEMPLATES
Each strands of the DNA molecules serves as a
template strand for the
synthesis of new Daughter strand.
A template is required to direct the addition
of the appropriate complementary nucleotide to
the newly synthesized DNA strand.

27. Enzymes
DNA polymerase
Topoisomerase

28. TOPOSIOMERASE
Two types type 1 and type II
Topoisomerase type I catalyze the relaxation of
Super coiled DNA by breaking just one strand of
DNA Where as type II cleaves both strands.
Topoisomerase II also called as DNA gyrase can
Introduce negative super coils to DNA using free
Energy from ATP hydrolysis.

29. PRIMER
The primer which is required for DNA synthesis
Is a short piece of RNA
( 10 nucleotides in length).
It is formed by DNA dependent RNA polymerase
Known as primase.
Primer is synthesized from 5’ to 3’ direction using
DNA as a template.

30. DNA polymerase initially adds a
deoxyribonucleotide to the 3’ hydroxyl group of
the primer and then Continues to add
deoxyribonucleotides to the 3’end of
the growing strand.

31. DNA POLYMERASE

32. • The DNA is copied by a replication
machine that travels along each
replication fork.
• One of the most important members
of this complex is DNA POLYMERASE

33. DNA Polymerase
• First discovered in 1956 by Kornberg
• Bacteria E.coli
• Bacteria have 3 types
– DNA Pol I, II, and III
• DNA Pol III involved in replication of DNA
• DNA Pol I involved in repair
Humans have 4 types
• DNA Pol alpha, beta, delta - nuclear DNA
• DNA Pol gamma - mitochondrial DNA

34. DNA Polymerase…
ALL DNA Pol’s have 2 properties
– Only synthesize DNA in one direction 5’
to 3’
– Only add to the end of existing double
stranded DNA
Therefore they CANNOT start synthesis
of DNA from scratch.
RNA polymerases can, but not DNA
polymerases

35. A comparison of prokaryotic and eukaryotic DNA
polymerase
Prokaryote Eukaryote Function
I α Primase activity, Gap filling
and synthesis of lagging strand
II ε DNA proof reading and repair
β DNA repair
γ Mitochondrial DNA synthesis
III δ Highly processive, leading
strand synthesis

36. ACTION OF DNA POLYMERASE I
After the completion of lagging strand synthesis
The RNA primers are removed from fragments
by DNA polymerase I.
The gap is filled by its polymerase activity.
The two polynucleotide chains are joined together
By another enzyme ligase.

37. DNA ligases:
The RNA primers must be removed and replaced by DNA.
After removal of the RNA segments and filling in the
Gap with DNA there remains points in DNA backbone
Where a phosphodiester bond is broken.
This is sealed by ligases.

38. ACTION OF DNA LIGASE
The joining of okazaki fragments is catalyzed
by DNA ligase.
This enzyme catalyses the formation of a
phosphodiester bond between the 3’ OH group at
The end of one DNA chain and 5’ phosphate
group at the end of the other.
This reaction requires energy

39. STEPS INVOLVED IN DNA REPLICATION
REPLICATION
1. Identification of the origins of replication.
2. Unwinding of dsDNA to provide as ssDNA
Template.
3.Formation of the replication fork.

40. 4.Initiation of DNA synthesis and
elongation.
5.Formation of replication bubbles
with ligation of the newly
synthesized DNA segments.
6.Reconstitution of chromatin
structure.

41. Protein Function
1 DNA
polymerase
Deoxynucleotide
polymerization
2 Helicases Progressive
unwinding of DNA
3 Topoisomerase Relieve torsional
strain that results
from helicase
induced unwinding.

42. 4 DNA primase Initiates synthesis of
RNA primers.
5 Single strand
binding protein
Prevents premature
rennealing of ds DNA
6 DNA ligase Seals the single
strand nick between
the nascent chain and
okasaki fragments of
lagging strand.

49. Fig. 3.8 Model for the “replication machine,” or replisome, the complex of key
replication proteins, with the DNA at the replication fork

50. STAGES OF REPLICATION
1.INITIATION
2.ELONGATION
3.TERMINATION

51. INITIATION
1.First DNA a protein recognises and binds to the
ori of the DNA and successfully Denatures the DNA.
and forms a replication bubble.
2. DNA B protein ( Helicase ) then binds to this region
and unwinds the parental DNA and forms a “ V “ where
active synthesis occurs. This is called as replication fork.
3. Topoisomerases releases the stress produced due to
super coiling by Helicase .
4. The SSB stabilizes the separated strands and
prevents their re association.

55. 5.Primase recognises a specific site on DNA
where RNA primer synthesis occur.

56. Elongation
DNA polymerase III complex assembles at the primer
sites and adds the corresponding bases based on the
template strand.
The DNA chain which runs in the 3’ to 5’ direction is
copied by polymerase III in the 5’ to 3’ direction as a
continuous strand , requiring one primer and this is called
as Leading strand.
The DNA chain which runs in the 5’ to 3’ direction is
copied as a discontinuous manner.
This Is called as lagging strand. These fragments are
called as okazaki fragment.