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.