2.3 Replication of Nucleic Acids

Polymerase enzymes catalyze the duplication of nucleic acids. All of the DNA polymerases can only catalyze the addition of additional nucleotides to an existing piece of DNA that has a free 3'-hydroxyl group (they are unable to start a new strand without a primer attached). This limitation imposes some special conditions on the way new DNA is made in cells. The reaction itself uses deoxynucleotide triphosphates as substrates. The triphosphate supplies the free energy to drive the reaction (figure). Note that the enzyme only will add the next nucleotide base onto a primer, and the base to be added must be able to hydrogen bond with the base exposed on the strand to be copied (the template strand). During the reaction, inorganic pyrophosphate is liberated and immediately hydrolyzed to inorganic phosphate, insuring that the reaction will be irreversible.

The reaction only proceeds in the 5' to 3' direction (new bases are added to the 3' hydroxyl of the preceding sugar.

Figure 6. DNA polymerase requirses a primer, a template stand and the four deoxy nucleotide triphosphate substrates.

What are some of the requirements for DNA replication starting with intact DNA? First of all, replication starts at special sequences of bases that define the origin of replication (indicated as ori in our models). Then we must begin to unwind the DNA to provide access for DNA polymerase and other needed enzymes and proteins. As the DNA unwinds, we see two fork shaped areas that are indeed the replication forks. They proceed in both directions down the double stranded DNA, and each must have its own set of enzymes. Remember that the two strands of DNA are running in opposite directions. One strand is ready to serve as a template for the 5' to 3' synthesis, but the other is in the wrong orientation. To solve this problem, RNA polymerase is used to synthesize a small stretch of RNA (Okazaki fragment, named for the scientist who discovered this mechanism) to provide a primer so that DNA polymerase may continue the chain. The RNA stretches are removed, filled in with DNA and sealed together with an enzyme called ligase. A more detailed representation of the replication fork complex is shown in Figure 7. Notice that proteins are needed to help unwind the DNA, which is tightly coiled, before the synthesis may continue. By looping one strand around (the one that needs the RNA start) we may visualize the replication complex as all occurring as one large aggregate at the replication fork.

Figure 7. A more complete represenation of DNA synthesis at a replication fork.