We now consider DNA polymerase III holoenzyme, the bacterial replication machine, in greater detail. The first clue to this enzyme's existence came in 1969 when Paula Delucia and John cairns isolated an E. coli mutant that lacked DNA polymerase I activity but continued to synthesize DNA and grow normally. This mutant was considered to be very remarkable at the time of its discovery because investigators had assumed that DNA polymerase I was the only polymerase required for DNA synthesis. The possibility that the mutant might have a low level of DNA polymerase I activity that allowed it to synthesize DNA was ruled out when an E. coli mutant with a deletion in polA (the structural gene for DNA polymerase I) was shown to also synthesize DNA.
The most likely explanation for the polA mutant is ability to synthesize DNA is that some other DNA polymerase is present and that enzyme is responsible for DNA synthesis. In support of this hypothesis, two new enzymes-DNA polymerase II and DNA polymerase Ⅲ-were detected in polA mutant extracts when gapped DNA (created by partial hydrolysis of nicked DNA with an exonuclease) was used as a template. The two new polymerases add nucleotides to the 3'-end of the primer chain in the order specified by the template chain, Neither enzyme had been detected in bacterial extracts before because DNA polymerase I is so active that it masks their activity.
The next task was to determine what role, if any the new DNA polymerases play in DNA replication. Once again, a genetic approach helped to provide the answer Mutants lacking DNA polymerase ∏ synthesize DNA normally indicating that this enzyme is not essential for bacterial DNA replication. In contrast, temperature-sensitive DNA polymerase III mutants replicate DNA at 30 ° C but not at 42 ° C, indicating that DNA polymerase Ⅲ is required for bacterial DNA synthesis.
Although genetic studies indicated that DNA polymerase Ⅲ plays an essential role in bacterial DNA synthesis, the purified enzyme had few of the properties expected of the replication enzyme. For instance, DNA polymerase Ⅲ could not extend a unique primer completely around a single-stranded circular DNA template even when large quantities of enzyme and substrate were added to reaction mixtures for long periods of time. Furthermore, DNA polymerase Ⅲ synthesized DNA at about 20 nucleotides · s-1, an exceptionally slow rate when compared to the 1000 nucleotides · s-1 observed in the living cell. The reason for this slow rate is that the enzyme dissociates from its DNA template rather frequently.
Enzymes that remain tightly associated with their template through many cycles of nucleotide addition are said to be highly processive. Processivity provides a quantitative measure of a polymerase's ability to remain tightly associated with its DNA template during DNA (or RNA) synthesis. A polymerase's processivity equals the average number of nucleotides the enzyme attaches to a growing chain before it dissociates from the DNA template.
