DNA polymerase I, it turns out, is not the main enzyme involved in replicating the bacterial chromosome. When the gene encoding the enzyme was mutated in E. coli, the bacteria were still able to replicate their chromosomes. They were, however, deficient in DNA repair. This suggested that DNA polymerase I is primarily involved in DNA repair (although it does play a role in replication, as we will see), and that another yet-to-be-discovered enzyme would be responsible for replication.

Eventually, two other DNA polymerases were identified, and named DNA polymerases II and III. These both had 5' to 3' polymerase activity like DNA polymerase I, and 3' to 5' exonuclease activity. Neither of these enzymes had the 5' to 3' exonuclease activity found in DNA polymerase I. The various enzyme activities of the different polymerases are summarized in the table below.

DNA polymerase III turns out to be the main enzyme involved in DNA replication. DNA polymerase II is a minor enzyme involved in DNA repair. DNA polymerase I is the main polymerase involved in DNA repair, and plays a specialized role in DNA replication, using its 5' to 3' exonuclease activity.

The Mechanism of Prokaryotic DNA Replication

As mentioned above, DNA replication in E. coli begins at OriC. It starts when the polypeptide products of the dnaA gene bind to the origin. These polypeptides cause localized strand separation. This allows a complex of the protein products of the dnaB and dnaC genes to bind. This complex acts as a helicase, which functions to unwind the DNA further. This unwinding produces the two replication forks. The unwound single-stranded region is kept single stranded through the action of single-strand binding proteins. There are other proteins that are found at the replication forks at this time, but their function is not well understood, so they will not be addressed.

At this point, a primer is needed so that DNA polymerase III can begin to act. As mentioned earlier, DNA synthesis needs a primer, so how is a primer produced? An enzyme called primase serves this purpose, by synthesizing a short stretch of RNA (generally from 5 to 15 nucleotides in length). RNA synthesis does not require a primer, so primase (which is a type of enzyme called an RNA polymerase) is able to synthesize a short primer where needed. Once this primer is made, DNA synthesis can begin, extending the polynucleotide chain originating with the RNA primer. Priming and synthesis occurs on both strands in the helicase complex moves along the parental DNA, shifting the replication fork, and allowing synthesis to continue.

This leads to another problem that has to be solved. As more DNA unwinds, and the replication fork moves along, synthesis of one strand (the lower strand in the diagram) can just continue, following the movement of the replication fork. The other strand being synthesized, however, cannot do this. As the replication fork moves along, it leaves a gap behind, as shown in panel B of the figure. To compensate for this, a second RNA primer must be synthesized a bit behind the first one, and DNA synthesized until it reaches the first primer (this is shown in panel C). You can easily imagine that as the replication fork progresses a bit further, this process will have to be repeated. Therefore, at each replication fork, the synthesis of one new DNA strand (the lower one in the figure) is continuous, while synthesis of the other strand must be accomplished in small increments, short stretch after short stretch; this type of synthesis is termed discontinuous. The strand of DNA that is synthesized continuously is called the leading strand, and the strand that is synthesized discontinuously is called the lagging strand. The small fragments of DNA making up the lagging strand are named Okazaki fragments, after the researcher who discovered them. Okazaki fragments are typically about 1000 to 2000 nucleotides each.

Discontinuous replication solves one problem but still leaves one matter unsettled: the lagging strand will be composed of individual, unjoined fragments of DNA and RNA. This is where DNA polymerase I comes into play. DNA polymerase I uses its 5' to 3' exonuclease activity to digest away the primer RNA, and replaces the primer with DNA by extending the strand from the adjacent Okazaki fragment. At this point all that is left to be done is to physically join the Okazaki fragments. This is accomplished by an enzyme known as DNA ligase. DNA ligase is able to join the 5' end of one DNA strand to the 3' end of another DNA strand.