
All cells undergo DNA replication and
cell division in order to give rise to a new
generation of cells
Mitosis- Division
of the nucleus of
a eukaryotic cell
into two
daughter nuclei
with identical sets
of chromosomes
Division of cell cytoplasm and organelles
of a cell into two daughter cells
 It is important that each daughter cell
has an exact copy of the parent cell’s
DNA

1958- Matthew Meselson and Frank Stahl
devised a clever experiment that suggested
that DNA replication is semiconservative
 They grew E. coli in a medium using
ammonium ions (NH4+) as the source of
nitrogen for DNA (as well as protein) synthesis
 They worked with two isotopes of nitrogen
 14N is the common isotope of nitrogen, but
they also used ammonium ions that were
enriched for a rare heavy isotope of nitrogen,
15N.

Semiconservative replication is the
process of replication in which each
DNA molecule is composed of one
parent strand and one newly synthesized
strand (daughter strand)
 Each daughter molecule receives one
strand from the parent molecule plus
one newly synthesized strand

Replication begins when proteins bind at
a specific site on the DNA known as the
replication origin
 In prokaryotes, the closed circular DNA
has only one origin of replication
 In eukaryotes, the linear DNA has
multiple origins of replication
 In both organisms, the two strands
forming the DNA molecule cannot simply
be pulled apart. Why?


The two strands of the
DNA molecule cannot
be simply pulled apart
because they are
held together by
hydrogen bonds that
are twisted around
each other to form a
double helix.
To expose a template strand, the two
parent DNA strands must be unravelled
and kept separate.
 DNA Helicase is a specific enzyme that
unwinds the double helix by breaking the
hydrogen bonds between the base pairs

DNA base pairs are
complementary to each
other and have a natural
tendency to anneal
 Anneal: the pairing of
complementary strands of
DNA through hydrogen
bonding
 Single-stranded binding
proteins (SSBs) are proteins
that keep separated strands
of DNA apart after DNA
helicase has unwound them

SSBs bind to the
exposed DNA single
strands and block
hydrogen bonding
The bacterial enzyme that relieves any
tension brought about by the unwinding
of the DNA strands during replication
 Gyrase works by cutting both strands of
DNA, allowing them to swivel around
one another, and then releasing the cut
strands
 Enzymes from the same family as gyrase
have similar functions in eukaryotes

DNA cannot be fully unwound because
of its large size compared with the size
of the cell
 The diameter of a human cell is ~ 5 μm
 The length of DNA is ~1 cm,
 That is a 2000-fold difference
 As a region of the DNA unwinds,
replication begins in two directions from
that origin

New complementary
strands are built as soon as
an area of the DNA has
been unwound
 Replication fork: As the
two strands of DNA are
disrupted, the junction
where they are still joined is
called the replication fork

In eukaryotes, more than one replication
fork may exist on a DNA molecule at
once because of the multiple sites of
origin
 This results in hundreds or replication forks
across a DNA strand and
 allows for rapid replication of DNA
 When two replication forks are quite
near each other, a replication bubble
forms

Origin of
Replication
Replication
Bubble
Replication
Fork
Parental
Strand
Daughter
Strand
Origin of Replication
Replication fork
Eventually, the replication
bubbles become
continuous and the two
new double-stranded
daughter molecules are
completely formed
Replication bubbles
In Prokaryotes, DNA Polymerase I, II, and
III are the three enzymes that function in
DNA replication and repair
 In Eukaryotes, five different types of DNA
Polymerase are at work






contains a chain of nucleotides
covalently linked together via
phosphodiester bonds
to form a sugar-phosphate backbone
with protruding nitrogenous bases.
A nucleotide consists of
 a nitrogenous base,
 a sugar, and
 a phosphate group

A nucleoside consists of a
nitrogenous base covalently
attached
 to a sugar (ribose or
deoxyribose)
 but without the phosphate
group
 Therefore, a nucleotide is a
“nucleoside-mono-phosphate”

DNA polyermerase III is the primary
enzyme responsible for replication.
 It's main function is to add the 5'
phosphate of a new nucleotide to and
existing 3'-OH group.
 Therefore, it synthesizes DNA in the 5' to 3'
direction, i.e., it adds free
deoxyribonucleoside triphosphates to a
3' end of an elongating strand

Why does it add deoxyribonucleoside
triphosphates ?
 The high energy of the triphosphates is
used to form bonds between the
nucleotides.
 The two outermost phosphates are
liberated, leaving the innermost group still
attached.
 Once the two outermost phosphates
have been released, the nucleoside
triphosphate has become a nucleotide

This enzyme has its own limitations
 It can't begin a new daughter strand by
itself - it requires that there already be a
3'-OH end to add the next nucleotide to
 Since DNA Polymerase III cannot initiate
a new complementary DNA by itself, an
RNA primer (10-60 base pairs of DNA) is
annealed to the template strand

The enzyme that builds RNA primers in a
5' to 3' direction since DNA polymerase III
cannot begin initiating on its own
 This primer provides the free 3'-OH
needed by DNA polymerase III.
 This initiation sequence is temporary and
is later removed and replaced by DNA
 Once in place, DNA Polymerase III can
start elongation by adding free
to the complementary strand

The first major step for the DNA
Replication is the breaking of hydrogen
bonds between bases of the two
antiparallel strands.
 The unwounding of the two strands is the
starting point.
 Helicase is the enzyme that splits the two
strands.

The initiation point
where the splitting starts
is called "origin of
replication"
 The structure that is
created is known as
Replication Fork


One of the most important
steps of DNA Replication is the
binding of RNA Primase in the
initiation point of the 3'-5'
parent chain

RNA Primase can attract RNA
nucleotides which bind to the
DNA nucleotides of the 3'-5'
strand due to the hydrogen
bonds between the bases.

When DNA is being replicated, two types
of daughter strand are being produced
Leading strand
 Lagging strand


Since DNA is always synthesized in the 5'
to 3' direction and the template strands run
antiparallel, only one strand is able to be built
continuously, that is the Leading strand.
The 3'-5' proceeding
leading strand uses a
5'-3‘ template
because DNA
Polymerase can
"read" the template
and continuously
adds nucleotides

The other strand is synthesized
discontinuously in short fragments in the
opposite direction to the replication fork
and is known as the Lagging strand
The 5'-3‘ lagging strand
uses a 3'-5‘ template
because DNA
Polymerase cannot
"read" the template and
continuously adds
nucleotides.
In the lagging strand the
RNA Primase adds more
RNA Primers.

Short fragments
of DNA that are
a result of the
synthesis of the
lagging strand
during DNA
replication

The enzyme that adds free
deoxyribonucleotides from primer to
primer forming Okazaki fragments

The enzyme that removes RNA primers
from the leading and lagging strands
and replaces the RNA primers with
appropriate deoxyribonucleotides

The enzyme that joins the
Okazaki fragments into
one strand by creation of
a phosphodiester bond

As the two strands of DNA
are synthesized, two
double-stranded DNA
molecules are produced
that automatically twist
into a helix