DNA polymerase I

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Overview

DNA polymerase I is an enzyme that mediates the process of DNA replication in prokaryotes. Pol I was the first enzyme discovered with polymerase activity, and it is the best characterized enzyme. Although this was the first enzyme to be discovered that had the required polymerase activities, it is not the primary enzyme involved with bacterial DNA replication. That enzyme is DNA Polymerase III (Pol III). It is 928 residues long, and an example of a processive enzyme - an enzyme which catalyzes a series of polymerisations. Discovered by Arthur Kornberg in 1956[1], 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:

  1. A 5' -> 3' DNA polymerase activity
  2. A 3' -> 5' exonuclease activity that mediates proofreading
  3. 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[2]. Cairns' lab assistant Paula De Lucia created thousands of cell free extracts from E.coli colonies and assayed them for DNA-polymerase activity. The 3,478th clone contained the polA mutant, which was named so by Cairns to credit "Paula" [De Lucia][3]. It was not until the discovery of DNA polymerase III that the main replicative DNA polymerase was finally identified.

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 only DNA polymerase 5' -> 3' activity, it is widely used in molecular biology.

Related Chapters

References

  1. Lehman, I. R. (1958). "Enzymatic Synthesis of Deoxyribonucleic Acid. I. Preparation of Substrates and Partial Purification of an Enzyme from Escherichia coli". J. Biol. Chem. 233 (1): 163–170. PMID 13563462. Unknown parameter |month= ignored (help); Unknown parameter |coauthors= ignored (help)
  2. Paula de Lucia (1969). "Isolation of an E. coli Strain with a Mutation affecting DNA Polymerase". Nature. 224 (5225): 1164–66. doi:10.1038/2241164a0. PMID 4902142. Unknown parameter |coauthors= ignored (help); Unknown parameter |month= ignored (help)
  3. Errol C. Friedberg (2006). "Timeline: The eureka enzyme: the discovery of DNA polymerase". Nature Reviews Molecular Cell Biology. 7 (2): 143–7. doi:10.1038/nrm1787. PMID 16493419. Unknown parameter |month= ignored (help)

Template:Polymerases

it:DNA polimerasi I


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