1. Replication of DNA
Chittranjan kumar
2019-335-010

2. DNA
Central dogma
RNA Protein
Transcription
Translation
By Crick

3. Key takeaways from Watson
and Crick model of DNA
1. Two adjacent nucleotides are
joined together by phosphodiester
bond.
2. Adenine always binds with Thymine
(uracil in case of RNA) and vice
versa.
3. Cytosine always bind with guanine
and vice versa.
4. Two strands run anti-parallel to
each other.

4. Replication
• Proposed by Watson and Crick.
• “ It has not escaped the notice
that the speci
f
ic pairing we
have postulated immediately
suggests a possible copying
mechanism for genetic
material’’ (watson and Crick,
1953)
• DNA is capable of self
duplication.
• Takes place in “S-phase” of the
cell cycle.

5. Semi conservative mode of DNA replication
• They grew E.coli in N^15 (heavy
isotope of nitrogen) as the only
nitrogen source for many
generations.
• E.coli (prokaryote)
• CsCl used as a density gradient.
• Light N^14 & heavy N^15 [NH4Cl]
• Result-In this experiment in
successive generation again heavy
DNA not found ,hybrid remains 2 in
number, and light DNA is
increasing due to N^14 medium.
Semiconservative mode of DNA replication was
f
irst proposed by Watson and crick.
Later it was experimentally proved by Meselson and Stahl (1958) in E-coli and Taylor
in vicia faba (1958)

7. Question:
After 60 minutes of replication, how many DNA will be present in the N^14 containing medium?
A) 6
B) 2
C) 8
D) cannot be predicted.
Question:
After 60 minutes of replication, out of total DNA, how many hybridDNA will be present in the N^14
containing medium?
A) 6
B) 2
C) 8
D) cannot be predicted.

9. Replication takes place…
Prokaryotes
1. Cytoplasm
2. Before during Binary
f
ission
Eukaryotes
1. In nucleus
2. S-phase

10. Origin of replication ‘Ori’
* A site from where replication starts.
* A-T rich site.
Prokaryote
* circular DNA.
* 1 ori.
* Unzipping starts at single
point.
Eukaryote
* More than 1 ori
* linear DNA
* Unzipping starts at multiple
points simultaneously.

11. Requirements
1. DNA {as template}
2. dNTPs [nuclleotides]- dATP,
dGTP, dCTP, dTTP

12. Enzymes involved
1. DNA dependent DNA polymerase: bind nucleotides by forming
phosphodiester bonds.
2. Primase [RNA polymerase]
3. Helicase- breaks the helix by breaking the H-bond.
4. Topoisomerase (in prokaryotes it’s called DNA-gyrase)- reduce supercoiling &
relieves tension.
5. DNA ligase
Proteins
1. SSB protein- Single Stranded DNA Binding proteins, prevents reformation
of H-bond.

13. DNA polymerase in prokaryotes
• DNA- polymerase-I :- this enzyme functions as exonuclease. It separates
RNA- primer from DNA and also
f
ills the gap. It is also known as DNA-
repair enzyme.
• DNA-polymerase-II :-it is least reactive in replication process. It is also
helpful in DNA-repairing in absence of DNA polymerase-I and DNA
polymerase-II
• DNA polymerase-III :- main enzyme of DNA- replication. It is most
important. The larger chains are formed by this enzyme. This is also
known as Replicase

14. Mechanism of DNA replication

15. UNZIPPING
• Starts at initiation point or
origin of replication.
• Enzyme responsible- Helicase.
• Mg+2 act as cofactor.
The separation of 2 chains of DNA is termed as unzipping

16. Formation of New chain

21. Important points of DNA replication

22. • Semi conservative
• Bidirectional
• Semi discontinuous
• Direction of polymerisation 5’-3’.
• Unzipping and polymerisation are simultaneous process .
• DNA replication is the process of polymerisation of nucleotide with
complementary base pairing.
• DNA polymerase can’t initiate the polymerisation as it requires pre-
existing 3’-OH (primer).
• dNPTs are present in nucleoplasm/cytoplasm.
• DNA replication is energy expensive process and dNTPs plays dual role.

23. • During replication if by mistake wrong nucleotide is incorporated , then
DNA polymerase itself remove the wrong nucleotide and adds the correct
one, this is called proof reading.
• End of choromosome (telomerase) is replicated by an enzyme telomerase
• Failure in cell division after DNA replication results into polyploidy.

24. Prokaryotic Replication Eukaryotic Replication
It is a continuous process. This process occurs in the S-phase of cell cycle.
Circular, double-stranded DNA Linear, double-stranded DNA with end
The DNA replicates in the cytoplasm The DNA replicates in the nucleus
Single origin of replication Multiple origins of replication
Small amount of DNA The DNA is 50 times more than prokaryotic DNA
DNA polymerase I and III are involved DNA polymerase α, β, γ, δ, and ε are involved.
Large okazaki fragments Small okazaki fragments
The process is rapid, 2000 base pairs per second The process is slow, 100 base pairs per second
Two circular chromosomes are obtained Two sister chromatids are obtained
DNA gyrase is required DNA gyrase is not required
Prokaryotic Replication vs Eukaryotic Replication
Following are the important difference between prokaryotic and eukaryotic replication: