DNA Replication

One of the most important properties of DNA functioning as the genetic material is that it can make exact copies of
itself (autocatalytic function) forming the basis for transmission of hereditary characters it controls. This process is
called replication.

Replication of a DNA molecule gives rise to two identical daughter molecules.

After division whole genome replication completed and transferred to daughter cells.

Semiconservative:

The semiconservative mode of DNA replication was suggested (1953) by Watson and Crick along with the double-
helix model of DNA.

Here the replication of DNA involves the progressive separation of the two strands of DNA molecules by breaking up
of hydrogen bonds between base pairs.

Each strand, acting as template, synthesizes their complementary new strand on itself taking raw materials from the
nuclear sap. Thus two daughter DNA helices are formed.

Each daughter DNA helix has one old or parental and one new strand.

Meselson-Stahl Experiment:

The experimental evidence for semiconservative replication of DNA was first demonstrated by Mathew Meselson
and Franklin Stahl in 1958.

They grew cells of the bacterium Escherichia coli on a medium that contained 15N (a heavy isotope of 14N) in the form
of ammonium chloride (NH4C1).

The cells of E. coli were grown for 14 cell generation so that the cells used the 15 N to synthesise bases which were
then incorporated into DNA.

DNA having 15N has a detectable higher density (1.724 gm/cm2) than that having 14N (1.710 gm/cm2).

Therefore, they are called heavy and light DNA, respectively. Heavy and light DNA can be separated readily through
equilibrium density gradient centrifugation where they form distinct band in the centrifuge tube

The cells of E. coli were then sub-cultured and grown to a medium containing normal 14 N.

The transfer of cell to medium containing normal 14 N was followed by extraction of DNA from cells after zero, one,
two, three etc. cell generations (one cell generation represents the time during which all the cells undergo one cell
division).

The extracted DNAs in each cell generation were analysed in cesium chloride (CsCl) density gradient.

DNA that containing 15N in both strand (heavy DNA) forms a band in the CsCl density gradient at a higher density
position than that contains 14N in both strand (light DNA).

After one generation of growth in 14N medium, the DNA bands at an intermediate (hybrid DNA) density.

Such hybrid DNA contains 15N in one strand and 14N in the other strand. After two generations of growth in 14N
medium, half of the DNA bands are seen at the hybrid density and half band at light density.

It may be pointed out that according to the conservative mode of replication, no intermediate band will appear after
one generation.
Only heavy and light bands would be formed. Similarly, the dispersive mode of replication would make only one
band having identical density. No light or intermediate bands would be formed.

Therefore, the result of Meselson and Stahl’s experiment clearly explains the semiconservative mode of DNA
replication, while the expectation of other mode of replication (conservative, dispersive) are not fulfilled.

Semiconservative mode of DNA replication in bacteria was also demonstrated by J.Cairns using autoradiography.

J.H.Taylor and his co-workers proved semiconservative mode of replication in Vicia faba (eukaryote).

Staining replicated chromosomes with fluorescent dye and Giemsa.The newly synthesized strand of each DNA stain
differently from old strand.

Chromosomes where the two strands of DNA are stained differently are called harlequin chromosomes.

Presence of harlequin chromosomes confirm semiconservative mode of replication.

Pre-requisites and steps of DNA replication

There are four basic components required to initiate and propagate DNA synthesis. They are: substrates, template,
primer and enzymes.

Substrates

Four deoxyribonucleotide triphosphates (dNTP's) are required for DNA synthesis. These are dATP, dGTP, dTTP and
dCTP. The high energy phosphate bond between the a and b phosphates is cleaved and the deoxynucleotide
monophosphate is incorporated into the new DNA strand.

Ribonucleoside triphosphates (NTP's) are also required to initiate and sustain DNA synthesis. NTP's are used in the
synthesis of RNA primers and ATP is used as an energy source for some of the enzymes needed to initiate and
sustain DNA synthesis at the replication fork.

Template
The nucleotide that is to be incorporated into the growing DNA chain is selected by base pairing with the template
strand of the DNA. The template is the DNA strand that is copied into a complementary strand of DNA.

Primer

The enzyme that synthesizes DNA, DNA polymerase, can only add nucleotides to an already existing strand or primer
of DNA or RNA that is base paired with the template.

Enzymes

Origin-binding Protein: binds and partially denatures the origin DNA while binding to another enzyme called
helicase.

Helicases: unwind double stranded DNA.

Single-stranded DNA Binding Protein (SSB): enhances the activity of the helicase and prevents the unwound DNA
from renaturing.

Primase: synthesize the RNA primers required for initiating leading and lagging strand synthesis.

DNA Polymerase: recognizes the RNA primers and extends them in the 5' to 3' direction.

Processivity Factors: help load the polymerase onto the primer-template while anchoring the polymerase to the
DNA.

Topoisomerase: removes the positive supercoils that form as the fork is unwound by the helicase.

RNaseH: removes RNA portions from Okazaki fragments.

Ligase: seals the nicks after filling in the gaps left by DNA polymerase.

An enzyme, DNA polymerase, is required for the covalent joining of the incoming nucleotide to the primer. To
actually initiate and sustain DNA replication requires many other proteins and enzymes which assemble into a large
complex called a replisome. It is thought that the DNA is spooled through the replisome and replicated as it passes
through.

In E.coli, the origin is a unique sequence of DNA of about 245 base pair long and known as Ori C. It is A-T rich and
recognized by a replication initiator protein which binds to the origin to begin replication.

In Yeast, the origin is known as Autonomous Replicating sequence (ARS) and is 150 base pair long. ARS is the binding
site for Origin Recognition Complex (ORC). The replication initiated from the origin proceeds along replication forks.
So, each origin has two terminii. One origin with its two unique termini is called a replicon.

The DNA replication can be unidirectional or bidirectional. At the origin when the two strands separate it forms a
replication eye.

DNA polymerase

This is the enzyme which polymerises deoxyribonucleotides. Arthur Kornberg and his colleagues in Washington
University in 1956 isolated the first DNA polymerase from E.coli. It was then known as Kornberg enzyme. other
polymerases like Polymerase II and polymerase III from E. coli. Polymerase III is the major polymerase involved in
DNA replication. Polymerase I and II are involved in DNA repair and proof reading in prokaryotes.
Mechanism of DNA Replication

The DNA double helix unwinds and uncoils into single strands of DNA by breakdown of weak hydrogen bonds.
Unwinding of helix is helped by enzyme helicases. Enzymes called topoisomerases cut and rejoin one strand of DNA
helping the separation of DNA helix.

If two highly inter-wined ropes are pulled apart by applying force, the two strands of ropes automatically inter-wine
as soon as application of force is stopped. And if one of the strands of inter-wined rope is cut, tension is relieved and
two strands fail to come together.

Enzymes like topoisomerases thus relieve the tension of DNA strands and two strands of helix are separated.
Actually, due to this unzipping of double stranded DNA replication bubbles are formed which subsequently extends
as a Y- shaped replication fork. In bacteria and many DNA phages, this extending is bi-directional.

Mechanism of DNA replication is the direct result of DNA double helical structure proposed by Watson and Crick. It is
a complex multistep process involving many enzymes.

1. Initiation:

It involves the origin of replication. Before the DNA synthesis begins, both the parental strands must unwind and
separate permanently into single stranded state. The synthesis of new daughter strands is initiated at the replication
fork. In fact, there are many start sites.

2. Elongation:

The next step involves the addition of new complementary strands. The choice of nucleotides to be added in the
new strand is dictated by the sequence of bases on the template strand. New nucleotides are added one by one to
the end of growing strand by an enzyme called DNA polymerase. There are four nucleotides, deoxyribrnucleotide
triphosphates dGTP, dCTP, dATP, dTTP present in the cytoplasm.

3. Termination:

All the end termination reactions occur. Duplicated DNA molecules are separated from one another.

The purpose of DNA replication is to create two daughter DNA molecules which are identical to the parent molecule.

Eukaryotic DNA Replication

The main polymerizing enzyme is polymerase a, b, g, d & e.

DNA pol III adds about 1000 nucleotides per second whereas DNA pol a adds about 50 nucleotides per second. The
SSB protein is known as Replication factor A in eukaryote and the topoisomerase is Type I topoisomerase.

eukaryotic DNA have multiple origin and each eukaryotic DNA is a multiple replicon.

During the S-phase of the cell cycle, the DNA replicates only once and then the cell divides.

in the anaphase stage the replication origins are licensed by a Replication Licensing Factors or RLF.

The RLF allows DNA to replicate once in the S-phase and the RLF get destroyed during replication.

Whole the process f DNA replication are same as that of prokaryotes.