LECTURE 2
DNA SYNTHESIS
(REPLICATION)
The replication process
• At each cell division the cell must replicate
(self duplicate) with extra ordinary accuracy.
• A cell must replicate its DNA before passing it
on to it descendants (daughter cells).
• During replication the parent DNA strand acts
template for its own duplication.
• The template strand specifies the sequence of
nucleotides on the complementary strand.
• For example, Adenine (A) will pair with
Thymine (T) and Guanine (G) will pair with
Cytosine (C).
• In each round of DNA replication, each of the
two strands of DNA is used as a template for
the formation of a new complementary strand.
• DNA replication is “semi-conservative”
because each daughter DNA double helix is
composed of one conserved strand and one
newly synthesized strand.
• DNA replication produces two complete
double helices from the original DNA
molecule, with each new DNA helix being
identical in nucleotide sequence to the original
DNA double helix
Mechanism of DNA replication
• DNA double helix is opened at replication
origins which is recognised by the initiator
protein.
• The initiator pulls apart the two strands of the
double helix structures forming two replication
forks.
• The opening up of the DNA double helix
structure attracts a group of enzymes that form
a replication machine, in which each enzyme
carries out a specific function.
• At each fork, a replication machine moves
along the DNA using each strand as a template
to make a new daughter strand.
• The two forks move away from the origin in
opposite directions, unzipping the DNA
double helix and replicating the DNA as they
go.
• DNA replication in bacterial and eukaryotic
chromosomes is therefore termed
bidirectional.
Role of different enzymes in DNA
replication
• For DNA replication to occur, the double helix
must be unzipped ahead of the replication fork
so that the incoming nucleoside triphosphates
can form base pairs with each template strand.
• Enzyme Topoisomerase unknots and uncoils the
DNA molecule to reduce the strain of unwinding
which would other wise impede the forward
movement of the replication machinery.
• Enzyme helicase break the hydrogen bonds to
unwind or unzip several sections of parent
DNA molecule.
• DNA-binding proteins cling to the single-
stranded DNA exposed by the helicase
preventing the strands from re-forming base
pairs and keeping them in an elongated form
so that they can serve as templates.
• DNA polymerase (Complex) extends both
the leading and lagging strand by catalysing
the addition of nucleotides to the 3ʹ end of a
growing DNA strand.
• The reaction involves the formation of a
phosphodiester bond between the 3ʹ end of
the DNA chain and the 5ʹ-phosphate group
of the incoming nucleotide.
• DNA polymerase does not dissociate from
the DNA each time it adds a new nucleotide
rather, it stays associated with the DNA and
moves along the template strand stepwise for
many cycles of the polymerization reaction.
• The template for the leading strand reads in a
3’ to 5’ direction where as the template
strand for the lagging strand reads in 5’ to 3’
direction.
• On the leading strand DNA polymerase III
requires enzyme primase (RNA polymerase)
to synthesise an initial RNA primer (short
piece of nucleotides, approximately 10
nucleotides long) for it to bind to before it
begins adding nucleotide in a 5’ to 3’ direction
continuously.
• For the leading strand, an RNA primer is
needed only to start replication at a
replication origin.
• On the lagging strand new primers are needed
continually as the movement of the replication
fork exposes a new stretch of unpaired bases
in the template strand.
• A new RNA primer is made at intervals along
the lagging strand.
• A Nuclease degrades the RNA primer and then
DNA polymerase I replaces RNA primers with
DNA nucleotides in a classic 5’ to 3’ away from
the replication fork until it runs into the next
RNA primer.
• DNA polymerase I forms short pieces called
okazaki fragments.
• The Okazaki fragments are joined by enzyme
DNA ligase which catalyzes the formation of
the phosphodiester bond by joining the 5ʹ-
phosphate end of one DNA fragment to the
adjacent 3ʹ-hydroxyl end of the next between
pieces of DNA.
• When the final RNA primer on the lagging
strand is removed, there is no way to replace it
.
• Therefore, the lagging strand would become
shorter with each round of DNA replication
leading to loss of valuable genetic information.
• Bacteria solve this “end-replication” problem
by having circular DNA
• Eukaryotes solve it by having long by
incorporating structures called telomerase to
the chromosome end.
• Enzyme telomerase extends the ends of the
replicating lagging strand by adding multiple
copies of the same short DNA sequence to the
template strand.
• This extended template allows replication of
the lagging strand to be completed by
conventional DNA replication.
END OF LECTURE!
THANK YOU.
