Although the semiconservative nature of DNA replication had been confirmed, many questions about replication remained. One of these questions was: is replication initiated at a specific site on the chromosome, or is it initiated at random sites, or even multiple sites?
The answer to this question depends somewhat on the organism being considered. Bacteria, for example, have a single specific origin of replication; in other words, bacterial replication begins at the same spot on the chromosome every time. In E. coli, this site is called OriC. OriC is a 9 base-pair (bp) sequence that is repeated four times within the region.
Eukaryotes also have specific sites at which replication is originated. However, because eukaryotic cells contain much more DNA than bacteria (humans have approximately 1500 times as much DNA as E.coli), there must be multiple origins of replication on each chromosome in order to replicate all of the DNA in a timely fashion. The amount of DNA replicated from a single origin is called a replicon.
Other research has revealed that DNA replication proceeds bidirectionally from an origin of replication. This means that replication proceeds in opposite directions away from the origin:
Note in the diagram how each original DNA molecule branches, or forks, at the point where replication is occurring. These branch points are called replication forks. Because replication is bi-directional, two replication forks form at each origin of replication. (Some rare examples have been seen where replication is unidirectional from the origin.) The open area of the chromosome between the replication forks is called a replication bubble.
DNA Polymerase I
DNA replication is catalyzed by a family of enzymes called DNA polymerases. The first of these enzymes to be discovered, DNA polymerase I, was isolated from bacteria (specifically, E. coli). Characterization of the activity of this enzyme in vitro revealed that it had certain requirements for activity. It needed 5'-triphosphate forms of the four nucleotides, and it required the presence of preexisting DNA. The DNA serves two purposes: 1) it serves as a template for the synthesis of the new DNA (the template determines the sequence of the new DNA strand, through the specificity of base pairing), and 2) it serves as a primer for DNA synthesis. It turns out that DNA polymerase I cannot initiate DNA synthesis without having a free 3'-OH to add a new nucleotide to. DNA synthesis therefore needs a primer, a preexisting piece of nucleic acid to serve as an initiator of DNA synthesis.
DNA polymerase I synthesizes DNA by forming a bond between the 5' phosphate of the incoming nucleotide (the other two phosphate groups from the nucleotide triphosphate are lost) and the 3' OH group of the nucleotide at the end of the growing DNA chain. If you draw this out for yourselves, you'll realize that this means the DNA chain being synthesized grows in a 5' to 3' direction. This is an important rule to remember: DNA polymerase synthesizes DNA only in a 5' to 3' direction.
In addition to its polymerase activity, DNA polymerase I has two other enzymatic activities, both of which are exonuclease activities. Exonucleases are enzymes that digest DNA (cleave phosphodiester bonds), chewing away at nucleotides from the end of the DNA chain. DNA polymerase I has 3' to 5' exonuclease activity, which degrades DNA in a direction opposite to that of synthesis. This provides the enzyme with a proofreading function: if a wrong nucleotide gets inserted into a growing chain, the enzyme can digest it out with the 3' to 5' exonuclease activity (almost like using the delete key while word processing), and insert the correct nucleotide.
DNA polymerase I also has a 5' to 3' exonuclease activity, which degrades nucleic acids in the same direction as synthesis. As we'll see, this activity is used to remove primers during DNA replication.