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.