The key enzymes involved in DNA replication are helicase, single-stranded DNA binding proteins, topoisomerase, DNA polymerase, DNA primase, and DNA ligase. Helicase unwinds the DNA double helix using ATP hydrolysis. Single-stranded DNA binding proteins bind to the separated strands and prevent them from reannealing. Topoisomerase controls the topology of the DNA duplex during replication. DNA polymerase synthesizes new DNA strands using the old strands as templates. DNA primase synthesizes RNA primers for DNA polymerase. And DNA ligase joins the DNA fragments produced during lagging strand synthesis.

1. Enzymology of DNA
replication

2. The enzymes that are involved in DNA replication –
DNA replication
enzymes
DNA
Helicase Single
stranded
DNA
binding
proteins
(SSP)
Topoisomerase
DNA
polymerase
DNA
primase
DNA
ligase

4. DNA Helicase
Helicase – used to separate strands of a DNA double helix or
self- annealed RNA molecule using the ATP hydrolysis
 The human genome codes for 95 non-redundant helicases:
64 RNA helicases and 31 DNA helicase
 DNA helicase – first discovered in E.coli (prokaryote)in
1976; the lily plant (eukaryote) in 1978
Role: “ unwinding of DNA helix during replication in a
ATP- dependent reaction ’’ - resulting in DNA replication
fork
It maintains 3000 resolution/minute
Depending on work – Classified as α
helicase (works on single strand ) & β
helicase (works on double strand)

5. Single stranded DNA-binding protein
(SSB)
 It binds to the single- stranded region of DNA
Role: During DNA replication these SSB binds to the newly
separated individual DNA strands , thereby it holds them in a
place so that each strand ca act as template for new DNA
synthesis
It prevents the intra-binding of nucleotides during DNA
loop formation (a process in DNA fork formation)
It is coded by “ssb gene” in E.coli
The most studied SSB protein is gp30

6. Topoisomerase
 The coling of DNA molecule can lead to changes in topology
, including positive or negative supercoils
This leads to the stress on DNA duplexes.
Role: Controls the topology of the DNA duplex during
replication
The first topoisomerase discovered was E.coli
topoisomerase I (Topo I)- removes negative supercoils without
leaving nicks in the DNA molecule
This enzyme usually acts on the single strand of the DNA and
creates Single stranded break

7. Topoisomerase II (Topo II) – was first discovered from E.coli
and named as DNA gyrase
It has the ability to cut both strands of a double-stranded
DNA molecule, pass the portion of the duplex through the cut
and reseal the cut in a process that utilizes ATP
DNA gyrase has 2 identical subunits
Role: 1) introduce negative supercoils at or near the OriC site
in DNA template
 2) to remove the postive supercoils that form ahead of the
growing fork during elongation
All type of Topo II catalyze the catenation and decatenation
(linking and unlinking) of two different DNA duplex
In E.coli decatenation is catalyzed by DNA gyrase and second
type II enzyme , called Topoisomerase IV (Topo IV)

8. Movement of the growing fork
during DNA replication induces
formation of positive supercoils in the
duplex DNA ahead of the fork
In order for extensive DNA
synthesis to proceed, the positive
supercoils must be removed
(relaxed). This can be accomplished
by E. coli DNA gyrase and by
eukaryotic type I and type II
topoisomerases

9. DNA polymerase
 In 1957, Arthur Kornberg isolated an enzyme from E.coli
called “ kornberg enzyme” during an attempt of in-vitro DNA
synthesis
It is now known as DNA Polymerase I
It is encoded by dnaA gene
He reported that DNA polymerase I
-has 5′ to 3′ polymerase activity (that requires free 5′
phosphate group and 3′ OH group
- requires primer with free 3′ OH group for action
-has 5′ to 3′ exonuclease activity
-has 3′ to 5′ endonuclease activity
 Later , it was known that DNA polymerase I cannot initiate
replication
As the DNA Pol II, III, IV, V was identified, DNA pol III is
known to be a true replicase

10. DNA polymerase III
Has 5′ to 3′ polymerase activity
Has 3′ to 5′ exonuclease activity
Has high accuracy – process called Proof reading
 Other DNA Pol involves in the DNA repair machanism
Eukaryote DNA Polymerase
 It is found to contain 5 types of Polymerase
1) DNA polymerase α (alpha)- relatively high molecular
weight enzyme – also called as cytoplasmic polymerase or large
polymerase (found in nucleus & cytoplasma)
2) DNA polymerase β (beta)- also called as nuclear
polymerase or small polymerase – found in vertebrates
3) DNA polymerase γ (gamma)- called as mitochondrial
polymerase and is encoded in nucleus
4) DNA polymerase δ (delta) - is found in mammalian
cells and is PCNA dependent for DNA- sythesis processivity

11. 5) DNA polymerase ε (epsilon) – known as DNA
polymerase δ II. This enzyme is PCNA independent
DNA primase
 Takes DNA as template and synthesis small RNA
primers to initiate DNA strand synthesis

12. DNA ligase
 It mediates the joining of fragmented DNA
 Usually the Okazaki fragments ( lagging strand) formed
during replication in 3′ to 5′ direction