DNA Replication
DNA replication is a complex cellular function that is necessary in order
to
sustain life and achieve growth. Many enzymes, proteins, and other
molecules
work together to ensure that genetic information is replicated
efficiently,
quickly, and accurately. Without any one of these components,
replication would
be very limited in its efficacy. DNA is comprised of two
strands of
complementary nitrogenous bases (adenine & thymine, guanine
& cytosine),
five-carbon sugars (either ribose or deoxyribose), and
phosphate groups. The
strands of DNA are arranged in a double-helix array and
are held together with
hydrogen bonds. The semiconservative replication model
is used to depict
replication. In this model, each new double helix has one
"old" strand
and one "new" strand. This is yet another way in which accuracy
is
ensured. Because the shape of the DNA molecule is extremely important to
its
functionality, care must be taken to ensure that all parts of the
molecule
remain in their appropriate space during replication, and that no
part of the
strand is broken. To replicate DNA, the two strands must first be
separated from
one another. The first enzyme used in this process is called
helicase. Helicases
use the energy from ATP molecules to unwind the
three-dimensional double helix.
While the strand is unwinding,
topoisomerase enzymes (such as gyrase) prevent
the strands from being winded
into a supercoil due to the torque produced by the
separating action. Since
each strand is comprised of complementary base pairs
that have a high
affinity to hydrogen-bond with one another, single-stranded
binding proteins
(SSBs) are attached to the strands to keep them from
reattaching to one
another. Once the strands are separated, work can begin to
construct two new
complementary strands that will ultimately attach to the
existing DNA strands
to form new complete DNA sequences. DNA polymerase III is
the active enzyme
that builds the new complementary strands. DNA polymerase III
is a
DNA-dependent enzyme. As such, a template (the existing separated
strand)
must be present to generate the new strand. DNA polymerase III
requires a primer
to begin its action. The primer used is a short RNA
sequence with a 3' hydroxyl
group that is formed by an enzyme known as
primase. This primer is usually about
ten nucleotides in length and is
complementary to the existing DNA strand. DNA
polymerase always works in the
same direction: from the 5' end to the 3' end.
Since DNA polymerase III
always works in the 5' to 3' direction, and DNA strands
are complementary,
this gives rise to a few minor issues that must be dealt
with. The strand in
which DNA polymerase can move in the same direction as
gyrase (with the
replication fork) is known as the leading strand. As the strand
is unwound,
DNA polymerase III can easily begin to replicate the strand, as
the
replication fork is already moving in the 5' to 3' direction. The
complementary
strand is known as the lagging strand. The replication fork is
necessarily
moving in the 3' to 5' direction on this strand. On this strand,
numerous primer
sequences are inserted so that the DNA polymerase III can
"backtrack"
to build the new sequence as the strand is unwound. The DNA
sequences between
these primers, which are 1000 to 2000 nucleotides long, are
known as Okazaki
fragments. Once DNA polymerase III has replicated the
fragments, the need arises
to remove the RNA primer sequences and fuse the
portions of the new strand
together. The first critical enzyme used to do
this is known as DNA polymerase
I. This enzyme removes the primer
sequence with the crucial 3' hydroxyl group
and synthesizes complementary DNA
to fill in the gaps left by the primers. After
this is completed, yet another
enzyme known as ligase is used to join the
fragments. This enzyme works by
forming a phosphodiester bond between the 3'
hydroxyl of the new strand and
the 5' phosphate group found on the Okazaki
fragment. Using each enzyme to
perform a specific function, DNA is successfully
replicated.