PROOF READING OF DNA POLYMERASE

 
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Proofreading of DNA polymerase HD

DNA Polymerase I (or Pol I) is an enzyme that participates in the process of DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase (and, indeed, the first known of any kind of polymerase). It was initially characterized in E. coli and is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene that encodes Pol I is known as polA. The E. coli form of the enzyme is composed of 928 amino acids, and is an example of a processive enzyme - it can sequentially catalyze multiple polymerisations.

Pol I possesses four enzymatic activities:

A 5' -> 3' (forward) DNA-Dependent DNA polymerase activity, requiring a 3' primer site and a template strand
A 3' -> 5' (reverse) exonuclease activity that mediates proofreading
A 5' -> 3' (forward) exonuclease activity mediating nick translation during DNA repair.
A 5' -> 3' (forward) RNA-Dependent DNA polymerase activity. Pol I operates on RNA templates with considerably lower efficiency (0.1–0.4%) than it does DNA templates, and this activity is probably of only limited biological significance.
In the replication process, RNAse H removes the RNA primer (created by Primase) from the lagging strand and then Polymerase I fills in the necessary nucleotides between the Okazaki fragments (see DNA replication) in 5' -> 3' direction, proofreading for mistakes as it goes. It is a template-dependent enzyme - it only adds nucleotides that correctly base pair with an existing DNA strand acting as a template. DNA Ligase then joins the various fragments together into a continuous strand of DNA.

Despite its early characterisation, it quickly became apparent that Polymerase I was not the enzyme responsible for most DNA synthesis — DNA replication in E. coli proceeds at approximately 1,000 nucleotides/second, while the rate of base pair synthesis by Polymerase I averages only between 10 and 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. Its role in replication was proven when, in 1969, John Cairns isolated a viable Polymerase I mutant that lacked the polymerase activity. 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 by Cairns to credit "Paula" [De Lucia]. It was not until the discovery of DNA polymerase III that the main replicative DNA polymerase was finally identified.