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Enzymology
of
DNA
Replication |
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Enzymology
of
DNA
Replication -
One
group
of
enzymes
involved
in
DNA
replication
which
have
been
studied
in
great
detail
are
the
DNA
dependent
DNA
polymerases.
At
least
three
are
known
which
catalyse
the
synthesis
of
a
complementary
structure
using
DNA
as
a
template.
The
first
of
these.
DNA
polymerase
I,
was
isolated
and
characterized
by
Arthur
Kornberg
for
which
he
was
awarded
the
Nobel
Prize
in
1959.
Till
the
early
sixties,
it
was
believed
that
this
was
the
only
enzyme
with
the
ability
to
catalyse
DNA
synthesis.
Later
in
the
sixties,
Roy
Curtiss
and
others
showed
that
bacterial
mutants
lacking
this
enzyme
still
had
the
ability
to
replicate
DNA
and
since
then,
two
other
DNA
polymerases
(DNA
polymerase
II
and
DNA
polymerase
III)
which
can
copy
DNA
have
been
recognized.
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In
addition
to
these
two
enzymes,
it
is
now
known
that
a
ligase
(joining
enzyme),
and
a
primase
(to
prime
the
synthesis),
in
addition
to
a
topoisomerase
and
other
replication
proteins
are
also
involved.
DNA
polymerase
I,
was
thought
to
be
the
major
enzyme
that
joins
together
deoxynucleotides
in
vivo
but
now
it
is
realized
that
DNA
polymerase
III
is
the
main
polymerizing
enzyme
while
DNA
polymerase
I
is
a
repair
enzyme
and
fills
In
the
gaps
between
the
small
fragments.
The
role
of
DNA
polymerase
II
is
not
yet
clear.
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All
these
three
known
polymerases
extend
the
DNA
molecules
from
the
5
end
to
the
3
end
only.
This
led
to
the
idea
that
the
replication
of
double
stranded
DNA
occurs
in
short
pieces
discontinuously
and
these
short
segments
are
later
joined
with
the
aid
of
a
ligase
to
form
a
continuous
strand.
Also, the DNA polymerases can add nucleotides only to a perfectly base paired nucleotide sequence and they cannot initiate new DNA synthesis. It now appears that the primary reaction is carried out by a DNA dependant RNA polymerase, which synthesizes short degradable RNA primers. It is also believed that in addition to the RNA polymerases, specific "primases", which differ from the RNA polymerases, synthesize the primary RNA which is then elongated by the DNA polymerase such a primase has been identified in the bacterium E. coli and also in the bacteriophages T4 and T7
Because of the difficulties in understanding the complex process of replication of bacterial double stranded DNA, investigators have used the single stranded DNA bacteriophage Øx174 as a test system to understand the exact mechanism of DNA replication.
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In this virus, a complementary strand (-) is first synthesised on the parental single strand (+ strand) to yield a double helical structure.
The new strand ( - strand) then serves as the template for the synthesis of more of + strands which are then incorporated into the viral particles. The process appears to be straight forward but is not as simple as stated. Discontinuous synthesis appears to occur even during this type of replication.
Evidence for discontinuous replication of DNA in bacteria first came from Okazaki in 1967, who was able to isolate short pieces of' DNA during replication. It now appears that not all replicating DNA molecules rely on RNA priming and Okazaki fragments to solve the problem posed by the properties of DNA polymerase. The involvement of t-RNA primers in the synthesis of DNA copies of tumor viruses has also been reported. Recently an explanation to describe the mechanism by which single stranded DNA in certain animal viruses replicates has been given.
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It is found that in single stranded DNA, "inverted repeats" (hair pin like structures) at each end of the molecule act as primers for initiation of replication.
The secondary structure of the DNA is a double stranded helix. For replication to occur semi conservatively, the structure must undergo unwinding. Over the years, a number of DNA binding proteins, (DNA destabilizing proteins and untwisting enzymes) have been found both in procaryotic and eucaryotic cells and are involved in opening up of the double stranded structure to allow replication. The latest addition to the list are the topoisomerases including the gyrase first isolated from E. coli. This new enzyme catalyses the introduction of negative super coils into double helical DNA in an ATP dependant reaction and appears to be essential for in vivo replication of DNA both in bacteria and bacteriophages. Antibiotics such as nalidixic acid and novobiocin inhibit this enzyme and there by prevent DNA replication at or beyond the replication point. It is believed to relieve the positive supercoiling strains which builds up during replication and aid in unwinding of the double helix.
One other point of interest yet to be solved with regard to DNA replication is the origin of replication and the number of replicating points per DNA molecule. Although a number of origins of replication have been sequenced the mechanism of control is not yet clear. It is however, generally agreed that a DNA molecule may have more than one replicating points from where replication can be initiated,The base sequence at the origin is not identical but has similarities. A variety of models have been described in literature to explain the mechanism of vivo DNA replication. Unfortunately, none of these provide a complete answer to the problem. The one that has received much attention an d is close to acceptance is the rolling circle model. According to this model, replication starts with a specific cut in one strand of the parental duplex molecule.
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This generates a terminal nucleotide with a free 3' OH group while the other end has a phosphate group at the 5' end. As replication proceeds, the 5' end of the open strand is rolled out as a free tail of increasing length. The replicating structure is called a rolling circle since the unraveling of the free single strand is accompanied by a rotation of the double helical template about its axis. The 5' end tail serves as a template for the synthesis of small DNA fragments which are eventually joined together by the DNA ligase. Such growing tails have a double stranded character soon after their formation. Elongation of such tails sometimes goes on to produce tails many times the length of the original circle. It is believed that the tails are cut by specific endonucleases and this is followed by circularization by pairing between the sticky ends
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