DNA Polymerase 19
- DNA Polymerase I (EC 2.7.7.7)
DNA polymerase I, Pol I, or Kornberg polymerase is the product of
the pol A gene of E. coli, which has now been cloned to yield an
overproducing strain.
2.1. Enzyme Data
The enzyme comprises a single polypeptide chain of mol mass
109,000 Da, with various enzymic activities. These are a 5'-3' poly-
merase, a 3'-5' exonuclease (proofreading activity), a 5'-3' exonu-
clease, and a phosphate-exchange activity. The polymerase activity
catalyzes the formation of a complementary DNA strand to an existing
single-stranded template, by the extension of a DNA primer possess-
ing a 3'-hydroxyl terminus. The latter may be provided merely by a
short gap or even a nick in the DNA duplex. The presence of these
multiple activities allows the use of DNA polymerase I to perform
several complex manipulations.
DNA polymerase I requires the presence of magnesium ions, as do
all DNA polymerases. (In some cases, manganese ions may substitute,
but reaction rates are lower, and there is a loss of fidelity.) The pH
optimum for the enzyme is 7.4, and sulfhydryl reagents are required.
Enzymic activity is measured by the polymerization of labeled nucle-
otides into double-stranded DNA using poly-dAdT as both template
and primer. One unit of activity catalyzes the incorporation of 10 nmol
into an acid precipitable form in 30 min at 37°C (4). DNA Polymerase
I can be obtained at a purity of 5000 U/mg.
2.2. Uses of DNA Polymerase I
There are several uses of DNA polymerase I in molecular biology.
First, it is used for the incorporation of labeled nucleotides into DNA
probes by nick translation (5). This requires the action of deoxyribo-
nuclease I (see Chapter 2) to introduce nicks into the duplex with
exposed 3'-hydroxyl termini to act as a substrate for the DNA poly-
merase I. 5'-3' Polymerization occurs concomitantly with 5'-3' exonu-
clease activity, which has the effect of physically translating the "nick"
along the DNA molecule and allowing the introduction of labeled
nucleotides into the newly replaced DNA strand. Reaction conditions
are such that one complete exchange of the DNA duplex is achieved