DNA polymerase I:
DNA Pol I posseses three
1. A 5' -> 3' DNA polymerase
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 (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.
DNA polymerase I obtained
from E. coli is used extensively for molecular biology research.
However, the 3' -> 5' proofreading activity is undesirable
for many in vitro molecular biology applications. However, this
activity can be removed by the proteolytic cleavage of the enzyme,
which releases a large peptide known as the Klenow fragment.
This fragment posesses only the polymerase and 5' -> 3' exonuclease