DNA replication is a complex process that involves unwinding of the DNA double helix, synthesis of new strands that are complementary to the original strands, and enzymes such as DNA polymerase and helicase. There are multiple origins of replication in eukaryotes that allow bidirectional replication from many starting points along DNA molecules. Enzymes involved include DNA polymerase alpha that works with primase to initiate DNA synthesis, and DNA polymerases delta and epsilon that carry out leading and lagging strand elongation. Telomeres prevent shortening of chromosomes with each round of replication through the action of telomerase.

1. DNA Replication
Dhanya. G
Lecturer
Dept., of Biotechnology
Mercy college, Palakkad

2.  Transfer of genetic information from parent to progeny
 Complex process
 Not completely understood
 DNA is autocatalytic and heterocatalytic
 Watson and Crick DNA model implies a mechanism for
replication:
a. Unwind the DNA molecule.
b. Separate the two strands.
c. Make a complementary copy for each strand.
INTRODUCTION

3. Basic rule for replication
• Nucleotide monomers are added one by one to the end of a
growing strand by DNA Polymerase enzyme
• Sequences in daughter strand is complimentary to the parent
strand
HOW THE 2 STRANDS OF A DAUGHTER MOLECULE ARE
RELATED TO THE 2 STRANDS OF PARENT MOLECULE???

4. Mode of replication

5. The Meselson - Stahl experiment

6. Origin of replication
• Site where replication begins
– 1 in E. coli
– 1,000s in human
• Strands are separated to allow replication machinery contact
with the DNA
– Many A-T base pairs, because easier to break 2 H-bonds
than 3 H-bonds

7. Replication fork

8. Direction of replication
• Unidirectional (eg:- replication of mt DNA in vertibrates,
prokaryotic DNA)
• Bidirectional (eg:- eukaryotic DNA)

9. Enzymes of DNA replication
 Nuclease
hydrolyse the phosphodiester bonds
• exonuclease
• endonuclease
 Polymerase or replicase
catalyse the formation of polymers

10. Prokaryotic DNA replication
RAW MATERIALS
 ENZYMES
 TEMPLATES
 RNA PRIMERS
 REPLICONS
 REPLISOMES
 PRIMOSOMES
o ENZYMES
 DNA polymerase (I, II, III)
 DNA helicases/ DnaB protein
 Topoisomerase/ gyrase
 SSBP

11. DNA polymerase I
• Discovered by Arthur Kornberg in 1956, it was the first known DNA
polymerase
• Composed of 928 aa
• Single 102KD polypeptide
• 3’ 5’ exonuclease activity, Proof reading
• 5’ 3’ endonuclease activity, nick translation during DNA repair
Functions:
1. Template site for binding template DNA
2. Primer site to bind primer RNA
3. Used to fill gap between Okazaki fragments that are formed
during lagging strand synthesis.
4. Catalyse DNA repair and discontinuous DNA synthesis

12. DNA polymerase II
• 90KD
• Coded by PolB gene
Functions:
1. Assist in polymerisation
2. Mainly involved in DNA repair
DNA polymerase III
• synthesizes base pairs at a rate of around 1000 nucleotides per
second
• Complex
• Act as 5’ 3’ and 3’ 5’ exonuclease
• Formed of 10 subunits
Functions:
1. Chain elongation in leading strand
2. Essential for in vivo DNA replication
3. Help in repair

13. DNA helicase/ Dna B protein
• Involved in strand separation
• ATP dependent enzyme
• 2 types
> Pri A protein
> Rep protein
Pri A protein
 or helicase II and III
 Moves on 5’ 3’
 Attaches to the template for the lagging strand
Rep protein
 Direct leading strand synthesis
 Moves on 3’ 5’
 In rolling circle replication

14. Topoisomerase or gyrase
• Causes topological changes in DNA
• Based on whether they cause single strand or double strand
break it is of 2 types
a> Type I topoisomerase
• Topo I and Topo III
• Temporary single strand breaks
• Relaxes negative supercoils
b> TypeII topoisomerase
• Topo II and Topo IV
• Break and reseal both strands

15. SSBP
• Single strand binding protein
• Tetramer
• Product of SSB gene
• No sequence specificity
• Helps in unwinding and prevent rewinding
o RNA PRIMER
• Short oligonucleotide to start replication
• Produced with the help of DNA primase/RNA polymerase
• Hydrogen bonded to DNA

16. o REPLICONS
• A discrete unit which helps in DNA replication
• In E.coli 1 replicon is present (OriC)
o REPLISOMES
• Carry out leading and lagging strand synthesis in a coordinated
manner
• complex molecular machine that carries out replication of DNA.
o PRIMOSOMES
• protein complex responsible for creating RNA primers on single
stranded DNA during DNA replication
• Consists of primase molecule linked to DNA helicase
• Moves along with replication fork and synthesis RNA primer
• DnaG primase, DnaB helicase, DnaC helicase assistant, DnaT, PriA, Pri B,
and PriC

17. Prokaryotic DNA replication

18. 1. Initiation
• DNA at the origin of replication denatures to expose the bases
• creating a replication fork.
• bidirectional
• one origin, oriC, which has:
a. A minimal sequence of about 245 bp required for initiation.
b. Three copies of a 13-bp AT-rich sequence.
c. Four copies of a 9-bp sequence.
Events
a. Initiator proteins DnaA attach.
b. DNA helicase (from dnaB) binds initiator proteins on the DNA, and
denatures the AT-rich region using ATP as an energy source.
c. DNA primase (from dnaG) binds helicase to form a primosome,
which synthesizes a short (5–10nt) RNA primer. .

20. 2. Elongation
Requires
• DnaB - Unwinding
• primase - primer addition
• DNA pol III - elongation
• SSBP - prevent rewinding
• RNAse H - removes RNA primer
• DNA pol I - fill the gap
• DNA ligase - join the okazaki fragments

21. • Discontinous synthesis of lagging strand
 Multiple primer needed
 RNA primer synthesized by primase
 Okazaki fragments are formed
 DNA pol I removes primer and adds nucleotides
 DNA ligase form phosphodiester bonds that link free 3’ end of
primer replacement of 5’ end of okazaki fragment.
• Continuous synthesis of leading strand
 Need only one primer
 RNA primer made by RNA pol
 DNA pol III for elongation in 5’--3’
 DNA pol I removes primer and adds nucleotides
 DNA ligase gives the final touch up

24. 3. Termination
• Occur at Ter site (7 identical non palindromic 23bp :Ter A, Ter
D, Ter E, Ter F and Ter G )
• Both clockwise( Ter G) and anti clock wise (Ter E)
• Some circular chromosomes (e.g., E. coli) are circular throughout
replication, creating a theta-like (θ) shape. As the strands
separate on one side of the circle, positive supercoils form
elsewhere in the molecule.
• Topoisomerases relieve the supercoils, allowing the DNA strands
to continue separating as the replication forks advance
Bidirectional replication of circular DNA molecules

25. Bidirectional replication of circular DNA molecules

26. Theta Replication
• Circular DNA in bacteria
• Replication bubble formed
from DNA unwinding and
strands separating
• Replication fork – point
where two strands separate
• Continues bi-directionally
until they meet

27. RollingCircle
Replication
• begins with a nick (single-stranded break) at the origin
• The 5’ end is displaced from the strand
• 3’ end acts as a primer for DNA polymerase III, which
synthesizes a continuous strand
• The 5’ end continues to be displaced as the circle “rolls”, and is
protected by SSBs until discontinuous DNA synthesis makes it a
dsDNA again
• During viral assembly it is cut into individual viral chromosomes
and packaged into phage head.

29. Eukaryotic DNA replication
• not as well understood as bacterial replication
• more complex
• Large linear chromosomes
• Tight packaging within nucleosomes
• More complicated cell cycle regulation
 In 1968, Huberman and Riggs provided evidence for the multiple
origins of replication
 DNA replication proceeds bidirectionally from many origins of
replication

30. ENZYMES

31.  DNA pol a is the only polymerase to associate with primase
 The DNA pol a/primase complex synthesizes a short RNA-
DNA hybrid
 10 RNA nucleotides followed by 20 to 30 DNA
nucleotides
 This is used by DNA pol d or e for the processive elongation
of the leading and lagging strands
 Current evidence suggests a greater role for DNA pol d
 The exchange of DNA pol a for d or e is called a polymerase
switch
 It occurs only after the RNA-DNA hybrid is made

33.  DNA polymerases also play a role in DNA repair
 DNA pol b is not involved in DNA replication
 It plays a role in base-excision repair
 Removal of incorrect bases from damaged DNA
 Recently, more DNA polymerases have been identified
 Lesion-replicating polymerases
 Involved in the replication of damaged DNA
 They can synthesize a complementary strand over the abnormal
region

34. Helicase
• 4 types
 Helicase A
 Helicase є
 Helicase ζ
 RF-A
2 types
 Class I ( Topo I & Topo III)
 Class II ( Topo II & Topo IV)
Human SSBP or RP-A
Tetramer
Topoisomerase
SSBP

35. Origins of Replication
• origins of replication found in eukaryotes have some similarities
to those of bacteria
 They are 100-150 bp in length
 They have a high percentage of A and T
 They have three or four copies of a specific sequence
 Similar to the bacterial DnaA boxes
 Origin recognition complex (ORC)
 A six-subunit complex that acts as the initiator of eukaryotic
DNA replication
 It appears to be found in all eukaryotes
 Requires ATP to bind ARS elements
 Single-stranded DNA stimulates ORC to hydrolyze ATP

36. Initiation
• Multiple origin
• Histones associated with DNA should be removed
• Rate 105 bp/min
• Takes 1000 times more replication time than that of prokaryotic
replication
• Large amount of DNA in chromosome at multiple replisomes
• Initiative protein selects the origin and activates it with the help of
other proteins
• Initiatve protein is ORC
• Y shaped intermediate will form

37. Elongation
• Primer excised by endonuclease or RNaseH
• Leading and lagging strand synthesis by the coupled action of
Polymerase and Helicase
• Histone reassociation after synthesis
• Ncleosome assembly
• Occurs by telomere replication
 Telomeric sequences consist of
 Moderately repetitive tandem arrays
 3’ overhang that is 12-16 nucleotides long
 Telomeric sequences typically consist of
 Several guanine nucleotides
 Often many thymine nucleotides
Termination

39.  DNA polymerases possess two unusual features
 1. They synthesize DNA only in the 5’ to 3’ direction
 2. They cannot initiate DNA synthesis
 These two features pose a problem at the 3’ end of linear chromosomes

40.  Therefore if this problem is not solved
 The linear chromosome becomes progressively shorter with
each round of DNA replication
 Indeed, the cell solves this problem by adding DNA sequences to
the ends of telomeres
 This requires a specialized mechanism catalyzed by the enzyme
telomerase
 Telomerase contains protein and RNA
 The RNA is complementary to the DNA sequence found in
the telomeric repeat
 This allows the telomerase to bind to the 3’ overhang

41. Step 1 = Binding
Step 3 = Translocation
The binding-
polymerization-
translocation cycle can
occurs many times
This greatly lengthens
one of the strands
The complementary
strand is made by primase,
DNA polymerase and ligase
RNA primer
Step 2 = Polymerization

42. http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.swf