Prokaryotic DNA polymerases | Chemistry of the Gene Synthesis, Modification and Repair of DNA | Genetics, Biotechnology, Molecular Biology, Botany

⇒	Chemistry of the Gene 2. Synthesis, Modification and Repair of DNA
⇒	DNA replication: general features
	⇒	Semi-conservative DNA replication in E. coli
	⇒	Semi-conservative replication of chromosomes in eukaryotes
	⇒	Semi-discontinuous DNA replication
	⇒	Unidirectional and bidirectional DNA replication
	⇒	RNA primers in DNA replication
	⇒	Regulation of DNA replication by anti-sense RNA primer
	⇒	Prokaryotic DNA polymerases
	⇒	Eukaryotic DNA polymerases
	⇒	Replicons for DNA replication
⇒	DNA replication in prokaryotes
	⇒	Experimental approaches for the study of DNA replication
	⇒	Initiation of DNA replication
	⇒	Elongation of DNA chain
	⇒	Replication fork movement
	⇒	Termination of DNA replication
⇒	DNA replication in eukaryotes
	⇒	DNA replication and cell cycle
	⇒	Replication origins and initiation of DNA replication (cis and trans-acting elements)
	⇒	Comparison of initiation of DNA replication with transcription initiation
	⇒	Different steps involved in eukaryotic DNA replication
	⇒	Synthesis of telomeric DNA by telomerase
⇒	Models of DNA replication
	⇒	Replication fork model
	⇒	Rolling circle model of DNA replication
	⇒	Mitochondrial DNA replication and D-loops
⇒	RNA directed DNA synthesis (reverse transcription)
⇒	DNA modification and DNA restriction
⇒	DNA repair
	⇒	Excision repair systems in E. coli
	⇒	An SOS repair system in E. coli
	⇒	DNA repair and genetic diseases in humans

Prokaryotic DNA polymerases
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.

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).

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).





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