Unit IV.

DNA replication
DNA Replication:
It is a complex process that occures during cell division, (interphase, S phase) whereby DNA makes copies
(duplicates) before the cell divides through mitosis and meiosis.DNA replication is an essential mechanism
in enhancing cell growth, repair, and reproduction of an organism.
DNA replication is a semiconservative process where a parental strand (template) is used to synthesize a
new complementary daughter strand using several protein elements which include enzymes and RNA
molecules.
DNA replication process uses DNA polymerase as the main enzyme for catalyzing the joining of
deoxyribonucleoside 5′-triphosphates (dNTPs) forming a growing chain of DNA.
Other proteins are also involved for initiation of the process and copying of DNA, along with proofreading
capabilities to ensure the replication process takes place accurately.
Therefore DNA replication is a process that produces identical helices of DNA from a single strand of the
DNA molecule.
Semi-conservative replication: In this model, the two strands of DNA unwind from each other, and each
acts as a template for making a new, complementary strand. This makes two molecules of DNA, one with
the old strand and one with the new strand.
Meselson and Stahl Experiment
Meselson and Stahl grew E.coli on a medium that contains 15NH4Cl as the only nitrogen source for many
generations [Note: 15N is the heavy isotope of nitrogen]. As a result, all newly synthesized DNA had 15N
which can be differentiated from normal DNA by centrifugation in a cesium chloride (CsCl) density
gradient.
They then transferred the cells to a medium containing normal 14NH4Cl.
As the cells multiplied, they collected samples at time intervals (20mins, 40mins, 60mins etc). This is
because E. coli cells divide every 20 minutes. They then extracted the double-stranded DNA from the
samples and separated them on CsCl gradients to measure the DNA densities.

Results
DNA extracted after 20 minutes (one generation) in the 14NH4Cl medium had an intermediate density. This
is because it contained one parental DNA strand with the heavy 15N and one new DNA strand with the light
14N to give 15N14N.
DNA extracted after 40 minutes (two generations) in the 14NH4Cl medium showed equal amounts of
intermediate density and light density. This is because it contained equal amounts of the hybrid 15N14N
DNA (intermediate) and 14N14N DNA (light).
Enzymes involved in replication
DNA Helicase enzyme
This is the enzyme that is involved in unwinding the double-helical structure of DNA allowing DNA
replication to commence.
It uses energy that is released during ATP hydrolysis, to break the hydrogen bond between the DNA bases
and separate the strands.
This forms two replication forks on each separated strand opening up in opposite directions.
At each replication fork, the parental DNA strand must unwind exposing new sections of single-stranded
templates.
The helicase enzyme accurately unwinds the strands while maintaining the topography on the DNA
molecule.
RNA primase enzyme
This is a type of RNA polymerase enzyme that is used to synthesize or generate RNA primers, which are
short RNA molecules that act as templates for the initiation of DNA replication.
DNA polymerase
DNA polymerases are enzymes used for the synthesis of DNA by adding nucleotide one by one to the
growing DNA chain. The enzyme incorporates complementary amino acids to the template strand.
DNA polymerase is found in both prokaryotic and eukaryotic cells. They both contain several different
DNA polymerases responsible for different functions in DNA replication and DNA repair mechanisms.
In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. DNA
pol-I is a key enzyme, which participates in DNA duplication, proofreading, editing, repair and removal of
RNA primers. DNA poly-II is solely involved in DNA repair and DNA pol-III is solely involved in the
polymerization process.The first polymerase activity was seen in E.coli, which was observed by Arthur
Kornberg in 1958 and he named it as E.coli DNA-pol I.

Five types of DNA polymerase are present in eukaryotes, namely DNA pol-α, pol-β, pol-Ƴ, Pol- δ and pol-
Ɛ.Their function is given in the following table
DNA ligase enzyme
This is the enzyme that joins DNA fragments together by forming phosphodiester bonds between
nucleotides.
Exonuclease
These are a group of enzymes that remove nucleotide bases from the end of a DNA chain.
Topoisomerase
This is the enzyme that solves the problem of the topological stress caused during unwinding.
They cut one or both strands of the DNA allowing the strand to move around each other to release tension
before it rejoins the ends.
And therefore, the enzyme catalysts the reversible breakage it causes by joining the broken
strands.Topoisomerase is also known as DNA gyrase in E. coli.
Telomerase
This is an enzyme found in eukaryotic cells that adds a specific sequence of DNA to the telomeres of
chromosomes after they divide, stabilizing the chromosomes over time.

Steps in replication
The semiconservative DNA replication process includes 3 steps.
 Initiation
 Elongation
 Termination
Initiation
This is the process that starts the process of DNA replication.
The enzyme helicase, which finds the ORI—Origin of replication, binds to the DNA strand and unwinds or
splits the double-stranded DNA molecule.
At the end of the step, the two strands of double-stranded DNA become two separate strands of DNA, but
not completely.
Half of the strand stays as a double strand because it doesn’t take part in the positive moment. This makes a
structure that looks like a fork, which is called the replication fork.
Elongation
This is where RNA nucleotides are added to the template DNA strand by the primer.
Another enzyme called Primase makes a short piece of RNA that the next enzyme finds and attaches to.
The part of the strand that goes from 3′ to 5′ is called the leading strand.
The part of the strand that goes from 5′ to 3′ is called the lagging strand.
Nucleotides are added by an enzyme called DNA polymerase. This enzyme adds the nucleotide that goes
with the template DNA.
The DNA polymerase can’t make a new strand from scratch; it can only add to one that’s already there.
In the end, enzymes called exonucleases will replace the RNA nucleotides with DNA nucleotides.
So, when there is adenine, thymine is added, and when there is guanine, cytosine is added.
Termination
Now, the process of making new strands must come to an end. DNA polymerase stops adding nucleotides
when the termination point is reached.
DNA polymerase can’t join the two pieces together because it can’t make a bond between them.
The enzyme ligase joins the strands together by adding a phospho-di-ester bond to the DNA molecule. This
brings the strands together.