DR VISHNU KUMAR

1. DNA Replication
DR. Vishnu Kumar
PROFESSOR & HEAD,
DEPT.OF BIOCHEMISTRY
MPTMC, SIDDHARTH NAGAR
COMPETENCY NUMBER BI 7.2

2. LEARNING OBJECTIVES
After completion of this lecture learner
should be able to define/ Describe:
• Important aspects of Replication
• Semi Conservative Replication
• Conservative Replication
• Meselson & Stahl Experiment
• Steps of Semi Conservative Replication
• DNA Polymerase

3. Replication
Every time a cell divides the entire content
of its chromosomal DNA must be duplicated
so that a complete complement can be
given to each daughter cell.
In this way the genetic information
is transmitted to new generation.
Replication may be defined as synthesis of
daughter DNA from mother DNA

4. Important aspects of replication
• Semi conservative.
• Starts at an A=T rich region called origin of
replication or ORI.
• Runs in 5’ to 3’ direction.
• Semi discontinuous.

5. Semi conservative replication
In replication two parental strands separate
and each strand serves as a template on
which a new strand is synthesized , so that
each daughter molecule contains one
parental strand and a new daughter strand.
The above process is called
Semi conservative.

10. Meselson & Stahl Experiment
The hypothesis of semi conservative
replication was proposed by Watson &
Crick and proved by Meselson & Stahl by
an experiment.
This experiment distinguishes between two
types of replication mechanisms.

12. The experiment
• Cells were grown for many generations in
a medium containing only 15N. So that all
nitrogen in the DNA were 15N.
• The cells were then transferred in a
medium containing only 14N for two
generations.
• Cellular DNA was isolated and centrifuged
to equilibrium in cscl medium.

13. Experiment continued..
• 15N DNA came to equilibrium at a lower
position than the 14N DNA.
• Hybrid DNA equilibrated at an
intermediate position.
• Conservative replication would yield 15N
DNA and 14n DNA and no hybrid DNA.

14. The prokaryotic replication
process
• The enzyme involved is DNA polymerase
that can synthesize the complementary
sequence of each strand with extra
ordinary fidelity.
• Prokaryotes contain DNAP I, II, & III.
• Eukaryotes have DNAP alpha, beta,
gamma, delta & epsilon.

15. Steps
• Initiation
• Separation of 2 strands & formation of
replication forks and replication bubble.
• RNA primer.
• Elongation
• Direction of replication.
• Excision of RNA primer & its replacement by
DNA.
• Termination.

16. Bidirectional replication

20. Proteins required for strand
separation
dna A protein20 – 50 monomers bind to
origin of replication which is an A=T rich
region. ATP required. This protein melts
the d/s DNA.
SSB binds to s/s of DNA and keep
them separated.
• Helicases force the two strands apart.
Needs ATP.

22. Problem of super coiling
• As the two strands separate the entire
chromosome ahead would accumulate
+ve super coils which will interfere with
further unwinding.
• Topoisomerases relax super coils during
replication and transcription. They perform
their role by cleaving phosphodiester bond
in one or both the strands. Types1-IV.

23. RNA Primer
• 10 to 60 nucleotide long RNA chain
synthesized by the enzyme primase at the
ORI on which new DNA strand will be
formed.
• Only one is synthesized in the leading
strand at the ORI but several primers are
formed in the lagging strand.

24. Elongation
• Elongation phase includes two distinct but
related operations.
• Leading strand synthesis.
• Lagging strand synthesis.

25. Elongation
• In the initiation phase the parent DNA is
first unwound by enzyme helicase and
topological stress is relieved by
topoisomerases. Each separated strand is
stabilized by SSB.
• From this point leading and lagging strand
synthesis are sharply different. Because
DNAPIII can synthesize new strand only in
5’ to 3’ direction not in 3’ to 5’ direction.

26. Leading strand synthesis
• Begins with synthesis by primase of a short (10-
60 nucleotide) RNA primer at ORI. DNTPs are
added to this primer by DNAPIII. Leading strand
synthesis proceeds continuously , keeping with
the unwinding of DNA at the replication fork.
Proof reading is also done.
• DNA strand is read by DNAPIII in 3’to5’ direction
and the new strand is formed in 5’to3’ direction.
Towards the replication fork.

27. Lagging strand synthesis
• Accomplished by short Okazaki fragments.
First an RNA primer is synthesized by
primase at the replication fork and as in
the leading strand DNTPs are added to
the RNA primer by RNAPIII.

28. Synthesis of Okazaki fragment
• Helicase and primase constitute a
functional unit within the replication
complex, the primosome.
• DNAPIII synthesizes the leading strand
continuously but it cycles from one
Okazaki fragment to the next on the
lagging strand.

29. Okazaki Fragment
• Helicase unwinds the DNA at the
replication fork.
• Primase component of primosome
synthesizes short RNA primer from time to
time. On each RNA primer DNAPIII adds
DNTPs.
• The entire complex responsible for DNA
synthesis at the replication fork is a
Replisome.

30. Removal of RNA primer &
replacement by DNA
• Once the Okazaki fragment has been
completed , its RNA primer is removed &
replaced with DNA by DNAPI & the nick is
sealed by ligases.
• As the new DNA strand encounters the
previous RNA primer, DNAPI comes and
removes the primer and replaces it by
DNA. Also proof reads.

31. Termination
• Two replication forks meet at a terminus
region containing multiple copies of a 20
base pair sequence called ’Ter” sequence.
‘Ter’ sequence binds with a protein called
‘Tus’ protein.
• When either replication fork encounters a
‘Ter-Tus’ complex it halts.
• separation of 2 strands by topoisomerase
IV.

32. Proteins at the replication fork
• SSB.
• Helicase.
• Primase.
• DNAPIII
• DNAPI
• DNA ligase.
• Topoisomerase II (DNA gyrase).

33. Eukaryotic replication
• DNA molecules in eukaryotic organisms
are considerably larger & are organized
into complex nucleoprotein structure,
chromatin. Essential features of replication
are the same in both the organisms, but
there are some variations.

34. Eukaryotic replication, Initiation
• Rate is slower, 1/20th that in E. coli.
• Origin of replication, called autonomously
replicating sequences (ARS) or replicators
have been identified.
• Initiation requires a multi subunit protein
the origin recognition complex (ORC),
which binds to several sequences within
the replicator.

35. Elongation
• As the rate is slower replication proceeds
bidirectionally from many origins.
• DNAP α,β,γ,δ & Є.
• Two other protein complexes, Replication
factor C (RFC) and replication protein A
(RPA) function .

37. Termination
• Involves synthesis of telomeres at the
ends of each chromosome.
• After replication there is reconstitution of
chromatin structure.

38. DNA Polymerase (S/N)
• The replication enzyme is DNAP. In
prokaryotes 3 types I, II, &III.
• DNAPI removes the RNA primer, replaces it
with DNA , also proof reads.
• DNAPII DNA repair.
• DNAPIII  main enzyme of replication. Adds
DNTPs to both the leading and lagging strand.
Also proof reads.

39. Eukaryotic DNAP
• DNAP α, β, γ, δ & Є.
• α primase activity, synthesizes short
primers for the Okazaki fragments on the
lagging strand. The primers are extended
by DNAP δ.
• β repair.
• γ  mitochondrial DNA synthesis.

40. Eukaryotic DNAP
–DNAP δ  multi subunit complex. 5’ 
3’ polymerase activity in both leading &
lagging strand., 3’  5’ exonuclease
activity (proof reading ). Associated with
proliferating cell nuclear antigen (PCNA)
which activates the enzyme.
• Є Removal of RNA primer from
Okazaki fragments, also in DNA repair,