King Abdulaziz University

Replicationof DNA

Ibraheem Ashankyty, PhD

Department of Medical Technology
Faculty of Applied Medical Sciences
 Unique Origins and
Bidirectional Replication
Objectives
Understand whether the origin of replication
is a unique site or occurs at random
differentiate between uni- and bidirectional
replication.
We now have direct evidence showing that
replication in E. coil and several other
organisms proceeds bidirectionally from a
unique origin.
Direction of DNA Replication
Origin of replication is the site at which
replication is initiated
Replication of DNA molecules in the
chromosomes of eukaryotes is
bidirectional.
Direction of DNA Replication
However, bidirection replication is not
universal. The chromosome of
coliphage P2, which like the lambda
chromosome is circular during
replication, replicates unidirectionally
from a unique origin.
SecondaryOrigins of DNA
Replication

It is clear that secondary unique site on

the T7 chromosome (normally inactive
in the presence of the primary origin
( becomes the active origin of
replication.
Requirement of Replication
DNA polymerase + MgCl2
Primer
DNA template chain
 DNA polymerase .1
(D(iscovery

The in vitro synthesis of DNA was first

accomplished by Arthur Kornberg and
his coworkers in 1957.
Konberg, who received the Nobel prize
in 1959 for this work, isolated an
enzyme from E.coli (initially called DNA
polymerase or “Kornberg enzyme,” now
known as DNA polymerase I).
 DNA polymerase .1
((Requirement of Replication

That catalyzes the covalent addition of

nucleotides to preexisting DNA chains
DNA polymerase requires MgCl2
Three different DNA polymenases (I,
II, and III)
In E. coil and B. subtilis, DNA
polymerase III is the major replicative
enzyme
 DNA polymerase .1
((3′5′ exonuclease activity

Most of the prokaryotic DNA polymerases

studied so far not only exhibit the 5′3′
polymerase activity, but also have a 3′5′
exonuclease activity.
(An exonuclease is an enzyme that
degrades nucleic acids from the ends, as
opposed to an endonuclease, which
degrades nucleic acids by making internal
cuts.)
 DNA polymerase .1
((Requirement of Replication

The 3′5′ exonuclease activity of DNA

polymerases carries out “proofreading”
or “editing function” that is necessary for
the high degree of fidelity characteristic
of DNA replication.
 DNA polymerase .1
((3′ 5′ exonuclease

When presented with a template-primer

DNA that has a terminal mismatch (an
unpaired or incorrectly paired base or
sequence of bases at the 3 ′ end of the
primer), the 3′5′ exonuclease of the
polymerase clips off the unpaired base
or bases.
 DNA polymerase .1
((Proof Reading

When an appropriately base-paired terminus

results, the polymerase begins resynthesis by
adding nucleotides to the 3′ end of the primer
strand and continues until the template is
exhausted.
This proofreading function is very important,
for DNA replication must be extremely
accurate.
Primer .2((Requirement of Replication
DNA. The DNA polymerase has an
absolute requirement for a free
3’-hydroxyl on a preexisting DNA
chain to form a phosphodiester bridge
between the 3’-OH at end of the
primer DNA chain and the
5’-phosphate of the incoming dNTP.
5’ to 3’ direction.
 .3 DNA templatechain
((Requirement of Replication
ssDNA template to which a primer is bound
DNA polymerase I does not contain sequence
specificity.
DNA template chain whose base sequence
dictates, via the DNA base-pairing
requirements, the synthesis of a
complementary base sequence in the strand
being synthesized.
 dNTPs .4
deoxyadenosine triphosphate (dATP),
deoxythymidine triphosphate (dTTP),
deoxyguanosine triphosphate (dGTP),
and
deoxycytidine triphosphate (dCTP)
 Watson-Crik Double
Helix Model
Diagram Space
filling
model
 DNA
Replication
As proposed by
Watson and
Crick
 E coli
Replication
Apparatus
Schem
DNA
replication
through
RNA
primers
that are
removed
by Pol I
 Polymerase III

Compositio
n of
polymerase
III
holoenzyme
of E. coli
 King Abdulaziz University

Semiconsevative
Replicationof
DNA
Ibraheem Ashankyty, PhD
Department of Medical
Technology
Faculty of Applied Medical
Sciences
 Meselson-Stahl
Experiment
Meselson and Stahl proposed that
DNA replicates semiconservatively
(1958)
The two other possibilities are:
• Conservative, or
• Dispersive
 Meselson and Stahl
grew E. coli for many generations in
medium that contains the heavy isotop
15
N substituting the light isotop 14N.
The DNA of cells grown in the heavy
isotop 15N will have a greater density
than DNA of cells grown in medium
containing the light isotop 14N.
 Meselson and Stahl
((Method
Took cells that had been growing in
medium containing 15N for several
generations washed them to remove
the 15n-containing medium, and
transferred them to medium containing
14
N.
Allowing the cells to grow in the
presence of 14N for varying periods of
time.
 Meselson and Stahl
((Method
The DNA was extracted and analyzed
in CsCl equilibrium-density gradients
using equiliprium dencity gradient
centrifugation technique.
6 M CsCl is which has a 1.7 g/cm3 was
used.
 Meselson and Stahl
((Method
The DNA containing 14N of E. coli has a
density of 1.710 g/cm3.
The DNA containing 15N has a density
of 1.724 g/cm3.
The equiliprium dencity gradient is
formed after centrifugation at high
speed 30,000 to 50,000 rpm for 48-72
hrs.
 Meselson and Stahl
((Results
The different DNA products were
separated on the bases of their density.
DNA will move to a position where the
density of the salt solution is equal to its
own density.
 Meselson and Stahl
((Results
Growth of bacteria in two different
growth medium resulted in two
different bands:
E. coil DNA containing 15N (“heavy”
DNA),
E. coli DNA containing 14N (“light”
DNA)
 After Centrifugation

E. coli DNA
containing 14N

E. coil DNA
containing 15N
 Meselson and Stahl
((Method 2
Bacteria grown previously in medium
containing 15N growth medium were
subjected to grow in a new growth
medium containing 14N for one
generation.
 Meselson and Stahl
((Result 2
The results was consistent with
semiconservative replication.
Excluding both conservative and
dispersive models of DNA synthesis.
 Meselson and Stahl
((Result 2
All the DNA isolated had a density
halfway between the densities of
“heavy” DNA and “light” DNA. (Referred
to as “hybrid” density)
 Meselson and Stahl
((Method 3
Bacteria grown previously in medium
containing 15N growth medium were
subjected to grow in a new growth
medium containing 14N for two
generation.
 Meselson and Stahl
((Result 3
After two generations of growth in
medium containing 14N, half of the DNA
was of “hybrid” density and half was
“light.”
As the No of grnrrations increases the
greater the ”light” band is.
 DNA After Different
Generations

Grown in14N Grown in14N Grown in14N Mixture

Grown (one (Two (Three Grown in14N
in15N (Generation (Generation (Generation and15N
((Control
 Conclusions
DNA replication is semiconservative
These results are precisely those
predicted by the Watson and crick.
 After Centrifugation

E. coli DNA
containing 14N
E. coli DNA
containing E. coil DNA
“Hybride” 14N containing 15N
and 15N
Thank You