DNA Replication

DNA Replication
• DNA replication is the process of producing two identical replicas from
one original DNA molecule.
• Copy genetic information for the transmission to the next generation
• Occurs in S phase of cell cycle
• Process of DNA duplicating itself
• Begins with the unwinding of the double helix to expose the bases in
each strand of DNA
• Each unpaired nucleotide will attract a complementary nucleotide from
the medium - will form base pairing via hydrogen bonding.
• Enzymes link the aligned nucleotides by phosphodiester bonds to form a
continuous strand.
 The mechanism of DNA replication

• During the replication process, the two

complementary strands of DNA come apart
and serve as template strands, or parental
strands, for the synthesis of two new strands of
DNA.
• After the double helix has separated, individual
nucleotides have access to the template
strands.
• Hydrogen bonding between individual
nucleotides and the template strands must
obey the AT/GC rule.
• To complete the replication process, a
covalent bond is formed between the
phosphate of one nucleotide and the sugar
of the previous nucleotide.
• The two newly made strands are referred to
as the daughter strands.
• The base sequences are identical in both
double-stranded molecules after replication.
• Therefore, DNA is replicated so that both
copies retain the same information—the
same base sequence as the original
molecule.
Models of DNA Replication

• Scientists in the late 1950s had considered three different mechanisms

to explain the net result of DNA replication.

• These are

1. Conservative model.

2. Semiconservative model.

3. Dispersive model.
Conservative model.
• According to this hypothesis, both
strands of parental DNA remain
together following DNA
replication.
• In this model, the original
arrangement of parental strands is
completely conserved, while the
two newly made daughter strands
also remain together following
replication.
Semiconservative model.

• In this mechanism, the double stranded

DNA is half conserved following the
replication process.

• In other words, the newly made double-

stranded DNA contains one parental
strand and one daughter strand.
Dispersive model

• Dispersive model, proposes that

segments of parental DNA and newly
made DNA are interspersed in both
strands following the replication
process.
 Messelson and Stahl experiment
• In 1958, Matthew Meselson and Franklin Stahl devised a method to
experimentally distinguish newly made daughter strands from the
original parental strands.
• Their technique involved labeling DNA with a heavy isotope of nitrogen.
• Nitrogen, which is found within the bases of DNA, occurs in both a
heavy (15N) and light (14N) form.
• Prior to their experiment, they grew Escherichia coli cells in the
presence of 15N for many generations.
• This produced a population of cells in which all of the DNA was heavy-
labeled.
The Meselson-Stahl experiment.
(a)Cells were grown for many generations in a
medium containing only heavy nitrogen,
15N, so that all the nitrogen in their DNA
was 15N, as shown by a single band (blue)
when centrifuged in a CsCl density gradient.
(b)Once the cells had been transferred to a
medium containing only light nitrogen, 14N,
cellular DNA isolated after one generation
equilibrated at a higher position in the
density gradient (purple band).
(c)Continuation of replication for a second
generation yielded two hybrid DNAs and
two light DNAs (red), confirming
semiconservative replication.
Unidirectional and Bidirectional replication
REPLICATION FORK:
• The leading strand is the strand of nascent DNA which is being
synthesized in the same direction as the growing replication fork.
• A polymerase "reads" the leading strand template and adds
complementary nucleotides to the nascent leading strand on a
continuous basis.
Unidirectional replication
• One fork moving away from the other which remains fixed at the origin
of replication.
Bidirectional replication
• Two replicating forks moving in opposite directions away from the
origin.
• Addition of tritium [3H] for a short period
just before the reaction is stopped allows
a distinction to be made between
unidirectional and bidirectional
replication, by determining whether
label (red) is found at one or both
replication forks in autoradiograms.

• This technique has revealed bidirectional

replication in E. coli, Bacillus subtilis, and
other bacteria.
Visualization of
bidirectional DNA
replication
• Replication of a circular
chromosome produces a
structure resembling the
Greek letter theta (θ).
• Labeling with tritium (3H)
shows that both strands are
replicated at the same time
(new strands shown in red).
• The electron micrographs
illustrate the replication of a
circular E. coli plasmid as
visualized by auto-
radiography.
Modes of replication

• There are mainly three modes of DNA replication mechanisms

• They are

1. Theta,

2. D-loop,

3. Rolling circle replication.

Theta replication
• The process of bacterial chromosomal replication is termed as theta
replication.
• The site on the bacterial chromosome where DNA synthesis begins is
known as the origin of replication.
• Bacterial chromosomes have a single origin of replication.
• The synthesis of new daughter strands is initiated within the origin
and proceeds in both directions, or bidirectionally, around the
bacterial chromosome.
Theta replication cont.….
• This means that two replication forks move in opposite directions
outward from the origin.
• A replication fork is the site where the parental DNA strands have
separated and new daughter strands are being made.
• Two replication forks can proceed independently around the DNA ring
and when viewed from above the structure resembles the Greek
letter "theta" (θ).
• Eventually, these replication forks meet each other on the opposite
side of the bacterial chromosome to complete the replication process.
Bacterial chromosome replication / Theta replication
Visualization of Theta replication
• A replicating E. coli chromosome visualized by autoradiography and
transmission electron microscopy (TEM).
• This chromosome was radiolabeled by growing bacterial cells in
media containing radiolabeled thymidine.
• The diagram at the right shows the locations of the two replication
forks.
• The chromosome is about one-third replicated.
• New strands are shown in blue.
D-loop replication /Displacement replication

• Some smaller circular molecules, such as animal mitochondrial genomes,

use a slightly different process called D-loop replication or displacement
replication.

• In these molecules, the point at which replication begins is marked by a D-

loop, a region of approximately 500 bp where the double helix is disrupted
by the presence of an RNA molecule base-paired to one of the DNA strands.
 D-loop replication cont.….
• This RNA molecule acts as the starting point for synthesis of one of the
daughter polynucleotides.

• This polynucleotide is synthesized by continuous copying of one strand of

the helix, the second strand being displaced and subsequently copied
after synthesis of the first daughter genome has been completed.

• The advantage of displacement replication as performed by animal

mitochondrial DNA is not clear.
D-loop replication
D-loop replication
D-loop replication
Rolling circle replication

• Rolling circle replication is an efficient mechanism for the rapid synthesis of

multiple copies of a circular genome.

• Rolling circle replication, which is used by λ and various other bacteriophages,

initiates at a nick which is made in one of the parent polynucleotides.

• The free 3’ end that results is extended, displacing the 5’ end of the
polynucleotide.
Rolling circle replication cont.….

• Continued DNA synthesis “rolls off” a complete copy of the genome, and
further synthesis eventually results in a series of genomes linked head to
tail.

• These genomes are single stranded and linear, but can easily be converted
to double-stranded circular molecules by complementary strand synthesis,
followed by cleavage at the junction points between genomes, and
circularization of the resulting segments.
Rolling circle replication
References
• Brooker, J. R. 2012. Genetics Analysis and Principle 4th Edition. Hill
International edition. McGraw.
• Fisher, M. (2001). Lehninger Principles of Biochemistry, ; By David L.
Nelson and Michael M. Cox. The Chemical Educator.
• Brown, T. A. (2007). Genomes 3. Garland Science Publishing
• Weaver, R. F. (2021). Molecular Biology, Fifth Edition. Hill International
edition. McGraw.