Bacterial DNA Polymerase I


Bacterial deoxyribonucleic acid (DNA) polymerase I is a family of enzymes involved in bacterial DNA synthesis and lesion repair. These enzymes have a multidomain structure, consisting of a single polypeptide chain that encompasses a distinct polymerase domain, a proofreading 3′–5′ exonuclease and/or a 5′–3′ exonuclease activity. Members of the polymerase I family have been investigated extensively, both because of their vital role in replicating and maintaining bacterial chromosomes, and because of their importance as tools in molecular biology, in particular for the polymerase chain reaction (PCR) and DNA sequencing. Studies on the Escherichia coliDNA polymerase I in particular have yielded significant insights into the mechanism of DNA polymerisation which is shared by almost all other nucleotide polymerases.

Key Concepts:

  • Bacterial DNA polymerase I enzymes are multidomain proteins with separate polymerisation, proofreading and 5′–3′ exonuclease functions.

  • They perform a vital function in bacteria by carrying out DNA repair and some aspects of chromosomal replication.

  • Polymerases incorporate one nucleotide at a time to the 3′ end of the primer DNA strand in a template‐directed manner.

  • Members of the DNA polymerase I family are able to replicate DNA with far greater accuracy than base pairing alone can account for.

  • The polymerisation reaction is a multistep process during which the enzyme switches between substrate binding and nucleotide incorporation conformations.

  • The 5′–3′ exonuclease allows the polymerase to degrade RNA primers and to assist with DNA repair.

  • Thermostable family I DNA polymerases are used extensively in polymerase chain reaction to amplify DNA and for DNA sequencing.

Keywords: DNA polymerase I; nuclease; kinetics; structure

Figure 1.

Structure of the Klenow fragment of Escherichia coli DNA polymerase I showing the large polymerase domain and the small 3′–5′ exonuclease domain. The N‐terminal exonuclease domain is shown in yellow, whereas the palm, thumb and fingers subdomains of the C‐terminal polymerase domain are coloured magenta, blue and green respectively. Helices are labelled with letters from A to R, whereas strands are labelled with numbers from 1 to 14. The division between the two domains is the loop between helices F and G (Ollis et al., ). This figure was produced using the coordinates with PDB entry code 1dpi.

Figure 2.

The open binary (a) and closed ternary (b) complexes of Klentaq with primer/template DNA and ddCTP. The N‐terminal small domain of Klentaq is shown in yellow; the thumb, palm and fingers subdomains of the large C‐terminal polymerase domain are shown in blue, magenta and green respectively, with the O‐helix in the fingers domain shown in red; the primer strand of the DNA is shown in cyan and the template strand is shown in orange, with the templating base highlighted in purple. The dCTP (shown in grey) in (a) is drawn to indicate the hypothetical dNTP‐binding site in the open complex; however, such an open ternary complex has not been captured in a crystal structure. Modified from Li et al. .

Figure 3.

The two metal ion mechanism for the catalysis at the polymerase active site. Modified from Steitz .



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Further Reading

Allen WJ, Rothwell PJ and Waksman G (2008) An intramolecular FRET system monitors fingers subdomain opening in Klentaq1. Protein Science 17: 401–408.

Bakhtina M, Roettger MP, Kumar S and Tsai MD (2007) A unified kinetic mechanism applicable to multiple polymerases. Biochemistry 46: 5463–5472.

Berdis AJ (2009) Mechanisms of DNA polymerases. Chemical Reviews 109: 2862–2879.

Bertram JG, Oertell K, Petruska J and Goodman MF (2010) DNA polymerase fidelity: comparing direct competition of right and wrong dNTP substrates with steady state and pre‐steady state kinetics. Biochemistry 49: 20–28.

Joyce CM, Potapova O, Delucia AM et al. (2008) Fingers‐closing and other rapid conformational changes in DNA polymerase I (Klenow fragment) and their role in nucleotide selectivity. Biochemistry 47: 6103–6116.

Rothwell PJ and Waksman G (2005) Structure and mechanism of DNA polymerases. Advances in Protein Chemistry 71: 401–440.

Rothwell PJ and Waksman G (2007) A pre‐equilibrium before nucleotide binding limits fingers subdomain closure by Klentaq1. Journal of Biological Chemistry 282: 28882–28892.

Tsai YC and Johnson KA (2006) A new paradigm for DNA polymerase specificity. Biochemistry 45: 9675–9687.

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How to Cite close
Allen, William J, Li, Ying, and Waksman, Gabriel(Sep 2010) Bacterial DNA Polymerase I. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001043.pub2]