Fig. 26.13. A model of DNA polymerase-I enzyme, showing five different sites, (redrawn from Kornberg, DNA synthesis - 1974).
Fig. 26.14. A model showing polymerization of nucleotides, on the primer site of DNA polymerase-I (redrawn from Kornberg, DNA synthesis - 1974).
Fig. 26.15. Removal of a mispaired nucleotide by exonuclease activity of DNA polymcrase I.
Fig. 26.16. Effect of four classes of enzymes on DNA duplex, showing cleavage in case of nucleases (endonuclease and exonuclease) and repair in the other two cases (redrawn from Watson, Molecular Biology of the Gene-1987).
Three different prokaryotic DNA polymerases
are known, of which DNA polymerases I and II are meant for DNA repair and DNA polymerase IN is meant for actual DNA replication, (i)
DNA polymerase I (isolated around 1960 by
Arthur Kornberg) was the first enzyme suggested to be involved in DNA replication. This enzyme is now considered to be a DNA repair enzyme rather
than a replication enzyme. It has five active sites shown in Figure 26.13 and its properties are shown in Table 26.1. This enzyme is mainly involved in removing RNA primers from
Okazaki fragments and fills
up the gaps due to its 5'→3' polymerising capacity. However, a ligase enzyme is needed for covalent bonding after the gap is filled by DNA polymerase I. It can also remove thymine dimers produced due to UV irradiation and fill the gap due to excision. Polymerising activity of DNA polymerase I is shown in Figure 26.14 and an exonuclease activity removing mispaired nucleotide is shown in Figure 26.15. This is described as
proofreading function of this enzyme. DNA polymerase I consists of two fragments : a larger fragment, called
Klenow fragment, which contains 3'-5' exonuclease activity with 5'-3' polymerising activity and a smaller fragment, which contains 5'-3' exonuclease activity. The activity of this enzyme (due to Klenow fragment) in using nicked DNA
in vitro is unique and is utilized to label DNA molecules with radioactive nucleotides.
This is called
nick translation, and is shown in Figure 26.16. DNA polymerase I enzyme is synthesized under the control of gene
polA located on
E. coli map at a position of 75 minutes, (ii)
DNA polymerase II resembles DNA polymerase I in its activity to bring about the growth in 5'→3' direction, using free 3'-OH groups, but mainly uses duplexes with short gaps only. It can not use nicked duplexes (unlike DNA polymerase I). Although it has 3'→5' exonuclease activity, it lacks 5’→3' exonuclease activity (exonuclease activity means cleavage of nucleotides only at the end, while endonuclease breaks DNA strand at an internal position). Since
pol B- mutants lacking DNA polymerase II appear normal in growth and conduct DNA replication normally, this enzyme can not be a replication enzyme. DNA polymerase II enzyme is also involved in DNA repair,
(iii)
DNA poiymerase III plays an essential role in DNA replication and is a heteromultimeric enzyme with ten units. All the ten subunits listed in Table 26.3 are needed for DNA replication
in vitro. However, the subunits have been divided into components of DNA replication system. For instance a subunit (coded by
polC or
dnaE)has 5'-3' synthetic activity and subunit e has 3'-5' exonucleolytic proofreading activity. The core enzyme, which has the ability to synthesize DNA, consists of subunits α
, β and θ. Other subunits increase the processivity (tendency to remain on a single template rather than to dissociate and reassociate again). Table 26.1 shows a comparison of DNA polymerase III with DNA polymerase I and DNA polymerase II of
E. coli. A comparison of synthetic activity of DNA polymerases with other related enzymatic activities (endonuclease, exonuclease, ligase) is presented in Figure 26.16.
Fig. 26.13. A model of DNA polymerase-I enzyme, showing five different sites, (redrawn from Kornberg, DNA synthesis - 1974).
Fig. 26.14. A model showing polymerization of nucleotides, on the primer site of DNA polymerase-I (redrawn from Kornberg, DNA synthesis - 1974).
Fig. 26.15. Removal of a mispaired nucleotide by exonuclease activity of DNA polymcrase I.
Fig. 26.16. Effect of four classes of enzymes on DNA duplex, showing cleavage in case of nucleases (endonuclease and exonuclease) and repair in the other two cases (redrawn from Watson, Molecular Biology of the Gene-1987).