DNA polymerase I
From Wikipedia, the free encyclopedia
DNA polymerase I is an enzyme that mediates the process of DNA replication in prokaryotes. Discovered by Arthur Kornberg in 1958, it was the first known DNA polymerase (and, indeed, the first known polymerase of any kind), and was initially characterized in E. coli, although it is ubiquitous in prokaryotes. It is often referred to as simply Pol I. In E. coli and many other bacteria, the gene which encodes Pol I is known as polA.
Pol I possesses three enzymatic activities:
- A 5' -> 3' DNA polymerase activity
- A 3' -> 5' exonuclease activity that mediates proofreading
- A 5' -> 3' exonuclease activity mediating nick translation during DNA repair.
DNA polymerase I removes the RNA primer from the lagging strand and fills in the necessary nucleotides of the Okazaki fragments (see DNA replication) in 5' -> 3' direction, proofreading for mistakes as it goes. It is a template-dependent enzyme, as it only adds nucleotides that correctly base pair with an existing DNA strand acting as a template. Ligase then joins the various fragments together into a continuous strand of DNA.
Despite its early characterisation, it quickly became apparent that Pol I was not the enzyme responsible for most DNA synthesis — E. coli DNA replication proceeds at approximately 1,000 nucleotides/second, while the rate of synthesis by pol I averages only 20 nucleotides/second. Moreover, its cellular abundance of approximately 400 molecules per cell did not correlate with the fact that there are typically only two replication forks in E. coli. Moreover, it is insufficiently processive to copy an entire genome, as it falls off after incorporating only 25-50 nucleotides. This was proven when, in 1969, John Cairns isolated a viable pol I mutant that lacked the polymerase activity. It was not until the discovery of DNA polymerase III that the main replicative DNA polymerase was finally identified.
[edit] Research applications
DNA polymerase I obtained from E. coli is used extensively for molecular biology research, however The 5' -> 3' exonuclease activity of E. coli's DNA polymerase I makes it unsuitable for many applications. Fortunately this undesirable enzymatic activity can be simply removed from the holoenzyme to leave a useful molecule called the Klenow fragment. Exposure of DNA polymerase I to the protease subtilisin cleaves the molecule into a small fragment, which retains the 5' -> 3' exonuclease activity, and a large piece called Klenow fragment. The large or Klenow fragment of DNA polymerase I has DNA polymerase 5' -> 3', and 3' -> 5' exonuclease activities, it is widely used in molecular biology.
