MOLECULAR BIOLOGY

DNA REPLICATION
Lecture 4
DNA replication
Before each cell division, new copies must be made of each of the
many molecules that form the cell, including the duplication of all
DNA molecules. DNA replication is the name given to this
duplication process, which enables an organism’s genetic information
— its genes — to be passed to the two daughter cells created when a
cell divides.

DNA replication is the process of producing two identical copies from

one original DNA molecule. This biological process occurs in
all living organisms . It is the basis for biological inheritance. DNA
is composed of two strands and each strand of the original DNA
molecule serves as template for the production of the complementary
strand, a process referred to as semiconservative replication.
After Watson and Crick proposed the double helix model of DNA,
three models for DNA replication were proposed: conservative,
semiconservative and dispersive.
Conservative Model
After DNA replication, the parental DNA
remains together, and the newly formed
daughter strands are together.

Semiconservative Model
The semi-conservative method suggests
that each of the two parental DNA strands
act as a template for new DNA to be
synthesized; after replication, each double-
stranded DNA includes one parental or “old”
strand and one “new” strand.

Dispersive Model
Both new copies of DNA have double-
stranded segments of parental DNA and
newly synthesized DNA interspersed.
How is semiconservative replication model discovered ?
Meselson–Stahl experiment :
An experiment by Matthew Meselson and Franklin Stahl in 1958, which
supported the hypothesis of DNA replication was semiconservative. It has
been called "the most beautiful experiment in biology. Each of the parent
strands will serve as a template to synthesis the complementary strand. The
following steps were performed to prove this theory:

Experiment
1-They cultured the bacterium E .coli in culture medium containing a
heavy Nitrogen ( N15) which is an isotope of nitrogen ( N14 ). (Since only
heavy nitrogen was the only source of Nitrogen for the bacteria they had to
use it during new DNA synthesis). Therefore their DNA would slowly be
replaced with DNA made of heavy nitrogen only. They maintained the
culture in a heavy nitrogen medium for several generations, they also
isolated the DNA of the heavy Nitrogen ( N15 ) grown bacteria and run
centrifuged on a salt density gradient. They got a single band of DNA that
had very high density.
2-Then, they sub-cultured those N15 containing E. coli on light
nitrogen ( N14) containing normal culture media. These bacteria can
now use only N14 as nitrogen source. Then they isolated the DNA
of E. coli after one round of replication and performed a centrifuged
on a salt density gradient. This time they got a single band of DNA in
the salt centrifuge tube. This band was higher, intermediate in density
between the heavy N15 DNA and the light N14 DNA.

The conservative model would have predicted two distinct bands in

this generation (a band for the heavy original molecule and a band
for the light, newly made molecule). This result fit with the
dispersive and semi-conservative models, but not with the
conservative models.
3-The second round of replication produced two bands of different
molecular weights. One DNA band was at the intermediate position
between N15 and N14, and the other band was higher (appeared to be
labeled only with the light N14). These results could only be explained
if DNA replicates in a semi-conservative manner. In contrast, in
dispersive replication, all the molecules should have bits of old and
new DNA, making it impossible to get a "purely light" molecule.
Therefore, the other two modes were ruled out
DNA replication process

the process of DNA replication followed by proofreading or error-

checking mechanisms to ensure correct reading of the genetic
code, like all biological polymerization processes (Transcription
and Translation, will be discussed later ), the process involve 3
stages :

1- Initiation 2-Elongation and 3- Termination

1- Initiation
The replication of both prokaryotic and eukaryotic DNAs starts at
a unique sequence called the origin of replication, which serves
as a specific binding site for proteins that initiate the replication
process.
In E. coli, which has a single origin of replication on its one
chromosome (as do most prokaryotes), it is approximately 245 base
pairs long and is rich in AT sequence ( rich in adenine and thymine
bases), because A-T base pairs have two hydrogen bonds (rather than
the three bond in a C-G pair) and easy to break in this site.

The origin of replication (oriC) is recognized by certain proteins that

bind to this site called Initiator proteins. These proteins (DnaA in
prokaryotes, origin recognition complex in yeast ) binds specifically
to the AT-rich replicator sequence oriC to form a specific DnaA-oriC
complex. An enzyme called helicase unwinds the DNA by breaking
the hydrogen bonds between the nitrogenous base pairs. ATP
hydrolysis is required for this process.
As the DNA opens up, Y-shaped structures called replication forks are formed. Two
replication forks are formed at the origin of replication and these get extended bi-
directionally as replication proceeds. (two replication forks begin at a single
replication origin in bacteria and proceed in opposite directions around the
chromosome forming θ theta shape, which look like a bubble , moving away from
the origin till reaching the opposite direction in one point called Ter Terminus (Teri).
http://biology-pictures.blogspot.com/2011/10/bidirectional-replication.html
The mechanism of eukaryotic DNA replication is similar to that of prokaryotic
DNA replication but it is more complex. There are multiple origins of replication
on the eukaryotic chromosome so multiple replication bubbles will form.

In yeast, which is a eukaryote, special sequences known as Autonomously

Replicating Sequences (ARS) are found on the chromosomes. These are equivalent
to the origin of replication in E. coli.
Single-strand binding proteins (SSBPs) bind to the single strands of
DNA near the replication fork to prevent the ssDNA strands from
winding back into a double helix,thus maintaining the strand
separation.
One of the key players in DNA replication is the enzyme DNA
polymerase, also known as DNA pol ( there are many types of DNA
polymerases in prokaryotes and eukaryotes will be discussed
later).
DNA polymerase is able to add nucleotides only in the 5' to 3' direction (a
new DNA strand can be only extended in this direction). It also requires a free
3'-OH group to which it can add nucleotides by forming a phosphodister bond
between the 3'-OH end and the 5' phosphate of the next nucleotide. This
essentially means that it cannot add nucleotides if a free 3'-OH group is not
available.
The problem is solved with the help of an RNA sequence that provides the
free 3′-OH end. RNA primase, synthesizes an RNA primer that is about five
to ten nucleotides long and complementary to the DNA template .
Because this sequence primes the DNA synthesis, it is appropriately
called the primer. DNA polymerase can now extend this RNA primer,
adding nucleotides one by one that are complementary to the template
strand. ( example: A in the template strand is complement to T in new
growing strand strand , and G in the template strand is complement to
C in new growing strand strand)… The primer is RNA rather than
DNA because DNA polymerases cannot start chains de novo
2- Elongation step

DNA double helix is anti-parallel; that is, one strand is in the 5' to 3'
direction and the other is oriented in the 3' to 5' direction. both
strands of parental DNA serve as templates for the synthesis of new
DNA. A new DNA strand is always synthesized in a 5’ to 3’
direction. Thus, the replication of both the strands goes in two
different ways .
One strand, which is complementary to the 3' to 5' parental DNA
strand, is synthesized continuously in 5----3 direction towards the
replication fork because the DNA polymerase III can add
nucleotides in this direction. This continuously synthesized strand is
known as the leading strand.
in prokaryote, DNA polymerase III begins the synthesis of the
leading strand by using the RNA primer formed by primase ( from
5’-3’direction or the same direction as the replication fork
movement) and add the nucleotides according to complement base-
paring to the template( A=T,T=A; G≅ C; C≅G)
However, all known DNA polymerases synthesize DNA in the 5′→3′
direction but not in the 3′ → 5′ direction.
How then does one of the daughter ( lagging ) DNA strands appear
to grow in the 3′→5′ direction?
The answer is
The other strand, complementary to the 5' to 3' parental DNA, is
extended away from the replication fork discontinuously; in small
fragments known as Okazaki fragments, each requiring a primer to
start the synthesis (this strand needs a new primer for each of the short
Okazaki fragments) . Okazaki fragments are then synthesized via
extension of these RNA primers by DNA polymerase. An important
consequence of such RNA priming is that newly synthesized Okazaki
fragments contain an RNA-DNA joint, the discovery of which provided
critical evidence for the role of RNA primers in DNA replication.
Okazaki fragments are named after the Japanese scientist Reiji Okazaki
(1968) who first discovered them. The strand with the Okazaki
fragments is known as the lagging strand.
Most current evidence indicates that DNA polymerases epsilon ε
and Delta δ, respectively, perform the bulk of leading and lagging
strand replication of the eukaryotic nuclear genome and Pol γ in the
mitochondria) .
As synthesis proceeds, the RNA primers are replaced by DNA. The
primers are removed by the exonuclease activity of DNA
polymerase I in prokaryotes, and the gaps are filled in by
deoxyribonucleotides. The nicks that remain between the newly
synthesized DNA (that replaced the RNA primer) and the previously
synthesized DNA are sealed by the enzyme DNA ligase that
catalyzes the formation of phosphodister linkage between the 3'-OH
end of one nucleotide and the 5' phosphate end of the other
fragment. ; this is the reason why the synthesis of the lagging strand
is more complicated than the leading strand.
A protein called the sliding clamp holds the DNA polymerase in
place as it continues to add nucleotides. (sliding-clamp proteins and
clamp-loading proteins) that load the polymerase onto the primer and
maintain its stable association with the template

in eukaryote, The enzyme ribonuclease H (RNase H), instead of a

DNA polymerase I, removes the RNA primer, which is then replaced
with DNA nucleotides. The gaps that remain are sealed by DNA
Ligase.
 3-Termina+on
1-Termination requires that the progress of the DNA replication
fork must stop or be blocked. Termination at a specific locus, when
it occurs, involves the interaction between two components: (1) a
termination site sequence in the DNA, and (2) a protein which
binds to this sequence to physically stop DNA replication. In
various bacterial species, this is named the DNA replication
terminus site-binding protein, or Ter protein.

2-Because bacteria have circular chromosomes, termination of

replication occurs when the two replication forks meet each other
on the opposite end of the parental chromosome . As a result, the
replication forks are constrained to always meet within the
termination region of the chromosome.
3-Removes the primer (RNA fragments), by 5'-3' exonuclease activity
of polymerase I, and replaces the RNA nucleotides with DNA
nucleotides. and fill the gaps.

4- When this is complete, a single nick on the leading strand and

several nicks on the lagging strand can be found. Ligase works to fill
these nicks in, thus completing the newly replicated DNA molecule .

6- Topoisomerase IV will : separate the two complete daughter

chromosome in to two chromosome.
To form a continuous lagging strand of DNA, the RNA primers must
eventually be removed from the Okazaki fragments and replaced with
DNA.
 Termination in Eukaryotic cell
vEukaryote cell initiate DNA replication at multiple points in the
chromosome, so replication forks meet and terminate at many
points in the chromosome; these are not known to be regulated in
any particular way. Because eukaryotes have linear chromosomes,
DNA replication is unable to reach the very end of the
chromosomes.
vPrimer removal at the end of the chromosome leaves a gap that
can’t be filled in (there is no DNA polymerase coming along to fill
in that piece. (Remember that DNA synthesis can ONLY occur
5’-3’). So on every round of replication, a little piece is lost from
the end of the chromosome.
Consequently, special mechanisms are required to replicate the
terminal sequences of the linear chromosomes of eukaryotic cells.
These terminal sequences sequences (telomeres) consist of tandem
repeats of simple-sequence DNA (short DNA sequences that are
repeated over and over at the ends of the chromosomes) Short
stretches are lost from telomeres at each round of replication. But
that’s alright because there is an enzyme called TELOMERASE
that can refill the telomeres from an RNA template (which is able
to maintain telomeres by catalyzing their synthesis in the
absence of a DNA template)
v Telomerase contains an RNA template to guide synthesis of new
telomeric repeats
 Enzymes involved in DNA replication

v Primase: in fact is RNA polymerase thus the formed primer is

RNA rather than DNA and it will removed latter by DNA
polymerase I
v Topoisomerase I: will break the 3́́ 5́ phosphodiester bond
converting super coiled to relax form which opposite to ligase.
Relaxes the DNA from its super-coiled nature

v DNA Helicase Also known as helix destabilizing enzyme

cases formation of Replication Fork due to broken hydrogen
bonds
v DNA Gyrase (and Topoisomerase IV) ; this is a specific type of topisomerase
II convert relaxed form to super coiled

v DNA Ligase Re-anneals the semi-conservative strands and joins Okazak’i

Fragments of the lagging strand.

v Telomerase Lengthens telomeric DNA by adding repetitive nucleotide

sequences to the ends of eukaryotic chromosomes

v DNA Polymerase Builds a new duplex DNA strand by adding nucleotides in

the 5' to 3' direction. performs proof-reading and error correction.

v DNA clamp: A protein (unit from polymerase which prevents DNA

polymerase III from dissociating from the DNA parent strand.

v Single-Strand Binding (SSB) Proteins Bind to ssDNA and prevent the DNA
double helix from re-annealing after DNA helicase unwinds it thus
maintaining the strand separation
Types of DNA polymerases in Prokaryotes
Named in order of discovery, DNA polymerase I, II, III
DNA polymerase III is the main polymerase for DNA replication (Main
enzyme that adds nucleotides in the 5'-3’) . DNA polymerase II is
involved in DNA repair.
DNA Polymerase I has :
1- Proofreading exonuclease in (3’à5’ direction )
2- Primer removal exonuclease in (5’à3’) direction
3-DNA synthesis (5’à3’) replaces primer with newly synthesized DNA

Types of enzyme Ini+a+on Polymeriza+on Exonuclease Exonuclease

ac+vity 5́́→3́ ac+vity 3́→5́ ac+vity
5́́→3́
DNA polymerase I
- + + +
DNA polymerase II
- + + -
DNA polymerase III
- + + -
Types of DNA polymerase in Eukaryotic cell
vThe DNA polymerases of eukaryotes are in general less understandable than the
DNA polymerases of prokaryotes. Eukaryotic cells have FIVE polymerases: four
major nuclear DNA polymerases: DNA polymerase alpha (Pol α), DNA
polymerase delta (Pol δ) and DNA polymerase epsilon (Pol ε), DNA polymerase
beta ( poly β), and one found in mitochondria: DNA polymerase Gamma (Poly
γ).

v1-Polymerase alpha ( Pol α ) : it is the only enzyme has primase activity beside
DNA polymerase, Pol α initiates DNA synthesis on both the leading and lagging
strands by synthesizing a RNA/DNA hybrid primer.

Pol α is a heterotetramer composed of two primase subunits and two polymerase

subunits. The primase subunits initiate DNA replication by synthesizing short (7–12
ribonucleotides) RNA primers, which are then extended by polymerase α.
v 2-Pol β Beta polymerase: excision repair and it is not highly
active and is not very processive.

v 3-Pol γ Gamma polymerase: polymerization the mitochondrial

DNA beside repairing by its exonuclease activity 3́→5́

v 4-Pol δ delta and 5-ε epsilon polymerase :polymerization

lagging (δ)and leading (ε) strand respectively 5 ́ → 3́ .
In eukaryotes.

v DNA can be synthesized in vitro by technique known as

Polymerase Chain Reaction (PCR) it will be discussed in
practical part of Molecular Biology course.