For MBBS, BDS, General Biochemistry students

1. Replication of DNA
Dr. V.P.Acharya

2. Hi! Let’s dive into the central dogma

3. CENTRAL DOGMA
The Flow of Information: DNA → RNA → Protein
A gene is expressed in two steps:
DNA is transcribed to RNA
Then RNA is translated into protein

4. DNA- major store of genetic
information
To transfer genetic information from
parent to daughter cell, DNA must be
duplicated.
DNA duplication– DNA
REPLICATION
DNA→DNA
 Semi- conservative replication

5. Different models of DNA replication

7. Salient features of replication
Each strand serves as a template/mould,
over which a new complementary strand is
synthesized
Base-pairing rule is maintained
Polymerization from 5’-3’ direction
Old DNA is not degraded but conserved
DNA polymerase synthesises a new
complementary strand

8. Requisites for replication
Substrates
4 deoxyribonucleotides; dNTP
Template
2 separated DNA strands
Enzymes
DNA polymerases
DNA topoisomerases
RNA Primer

9. Steps of DNA replication
Identification of origin of replication
Unwinding of dsDNA to provide an ssDNA
template
Formation of replication fork
Initiation of DNA synthesis and elongation
Formation of replication bubbles with
ligation of the newly synthesised DNA
segments
Reconstitution of chromatin structure

10. 3 stages of replication
Initiation
Elongation
Termination

11. Initiation
Replication starts with recognition of ori
(origin of replication)
Single site of ori– bacteria
Multiple sites– mammals
dnaA protein binds to ori
↓
Local denaturation & unwinding of an
adjacent A-T rich region

13. dnaB / Helicase– Binds to ori and unwinds
& separates the strands using ATP
↓
Replication fork formed
Topoisomerases– Relieve supercoils
Type I– Breaks one strand of DNA
Type II– Breaks both strands
Introduces negative supercoils
SSB (Single strand binding protein)-
Stabilizes the separated strands and
prevents reannealing.

14. Mammals- SSB counterpart is RFA
(replication factor)
dnaC protein– reqd. for dnaB binding
at ori
dnaG / Primase– Complexes with
proteins to form PRIMOSOME
↓
RNA primer synthesis

15. RNA Primer

16. RNA primer
10-200 nucleotides long
Primase in prokaryotes
DNAP α- eukaryotes
Nucleophilic attack by 3’- OH group of the
RNA primer on the phosphate of the first
entering deoxynucleotide triphosphate

17. Action of Helicase
Uses energy from ATP to
unwind the duplex DNA
SSB
SSB SSB
SSB

18. Replication fork

19. Components of replication fork
DNA helicase
Primase
DNA polymerase
SSB

20. Elongation
After RNA primers laid down, 2 DNA
polymerase III complexes attach
Leading strand & lagging strand
DNA chain which runs in 3’-5’ direction
copied by DNAP III in 5’-3’ direction in a
continuous manner
Okazaki fragments on lagging strand
DNAP I– removes RNA primers and fills in
the gap between okazaki fragments
Ligase– joins the segments

22. Okazaki fragments
Short stretches of 150-250bp (eukaryotes)
Found on lagging strand during DNA
replication
Later gap is filled by DNAP I and joined by
DNA ligase enzymes
Found in both eukaryotes and prokaryotes
Approx. 250 per replication fork

23. DNA polymerase complex
3 bacterial and 5 eukaryotic DNAP

24. Properties of DNAP
Chain elongation– 100
nucleotides/sec in mammals
Proof reading-- rectification
Processivity– How many nucleotides
are to be added before it dislodges

25. Proteins involved in replication

26. Termination

27. ter binding protein – binds to the sequence
↓
prevents helicase from
further action
↓
termination of replication

28. Which factor triggers DNA
replication???
External signals are delivered to cells
during the G1 phase of the cell cycle and
activate the synthesis of cyclins
Cyclins form complexes with cyclin-
dependent kinases (CDK)
Cascade of reaction
Synthesis of S phase proteins like DNAP
and thymidylate synthase

29. Eukaryotic replication
Binding of origin recognition complex(ORC)
to origins of replication during G1 phase
↓
ORC serves as a platform for highly
complicated pre-RC complex formation
↓
Converted to RC by CDK and Dbf4-
dependent kinase
↓
DNA polymerase α- primase
complex synthesizes first primer

30. 2 characteristic features
Histone complexes
Telomeres- repeated end sequences of
(TTAGGG)n and have typical sizes of 15–20 kb
at birth
↓
In somatic cells it is shortened after each cycle
↓
Germline and cancer cells have telomerases
↓
extend the 5′ end of lagging strands

31. Telomerase
Reverse transcriptase
Contains an RNA template
Telomerase over-activity- cancer
Under-activity- ageing

32. Modification after replication
Methylation of DNA
At C5 of C
Catalysed by DNA methyl transferase
Methylation occurs at G-C rich region of
promoter sequence
>90% methyl C s are in CpG dinucleotides
Methylated areas- transcriptionally silent
Aberrant methylation- cancer, ageing, ROS
dependent damage

33. Difference between prokaryotic and eukaryotic
replication
Features Prokaryotes Eukaryotes
RNA primer
length
~50 nucleotides 9 nucleotides
DNAP 3; I,II,III 5; α,β,γ,ε,δ
Number of
origin
Single Many
Okazaki
fragments
1000-2000
nucleotides
~250
nucleotides
Rate of
replication
~500
nucleotides/sec
~100
nucleotides/sec

34. DNA replicates once and once only
Pre-replicative sequence is formed-
ORC-DNA complex with Cdc6 and Cdt1 at
G1-S phase
New nucleosomes are assembled behind
the replication fork

35. Inhibitors of DNA replication
Topoisomerase II (DNA gyrase) inhibitor
Novobiocin– prevents ATP binding to gyrase
Nalidixic acid & Ciprofloxacin- interfere with the
breakage and rejoining of DNA chains
Camptothecin– inhibits human topoisomerase I
Nucleotide analogues– 2,3 dioxyinosine,
Cytarabine
Zidovudine, Acyclovir- terminate DNA chain
elongation

36. Be like the stem cell-differentiate yourself
from others

37. WWW.VPACHARYA.COM
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