18 MaundersThe choice of enzyme depends on the reaction conditions employed
and the desirability of other inherent enzymatic activities. The range
of enzymes available is rapidly expanding, ensuring a concomitant
increase in the applications of DNA polymerases in molecular biology.1.1. Prokaryotic DNA Polymerases
The classical E. coli DNA polymerase family consists ofDNA poly-
merases I, II, and III (Pol I, Pol II, and Pol III). Pol III is largely
responsible for chromosomal replication, and it initiates the growth of
both DNA chains on RNA primers. Pol I "fills in" the gaps left by the
intermittent nature of Pol III activity on the 3'-5' strand. Apart from
this activity, the major role of Pol I is in DNA repair rather than rep-
lication. The role of Pol II is still largely unclear.
The three enzymes have widely differing properties. Pol I, which is
the most ~videly used in molecular biology, is described in the next
section. Pol II has a polymerase activity of only 5% that of Pol I and
has no 5'-3' exonuclease activity. It can only act on duplexes with gaps
of < 100 bases and cannot replicate long single-stranded stretches from
short primers. Pol III has a similar template requirement to Pol II, but
is 300 times more active. Pol III holoenzyme consists of at least 13
components (1), although various smaller complexes are active in vitro.
In addition to the E. coli enzymes, DNA polymerases from other
bacteria are now attaining more prominence in molecular biology,
particularly those from thermophilic organisms. Other useful DNA
polymerases have been isolated from bacteriophage-infected E. coli
cells, especially the enzymes encoded by bacteriophages T4 and T7.
1.2. Eukaryotic DNA Polymerases
Eukaryotic organisms possess three main DNApolymerases, Pol a,
Po113, and Pol Y. Pol a is considered to be the major DNA polymerase
(2) and is responsible for chromosomal replication. The function of
Po113 is unclear, whereas Pol ?, being located in both the nucleus and
the mitochondria, is believed to be involved in mitochondrial DNA
replication. The three enzymes all lack 3'-5' exonuclease activity, which
has since been detected on a fourth DNA polymerase, Pol 8 (3). This
finding supports the idea that eukaryotic DNA polymerases work in
concert during DNA replication.