AIMS Biophysics, 2018, 5(2): 144-154. doi: 10.3934/biophy.2018.2.144

Research article

Export file:


  • RIS(for EndNote,Reference Manager,ProCite)
  • BibTex
  • Text


  • Citation Only
  • Citation and Abstract

Single-molecule FRET reveals proofreading complexes in the large fragment of Bacillus stearothermophilus DNA polymerase I

Konigsberg Laboratory, Yale University, 333 Cedar Street, New Haven, CT 06520, USA

There is increasing interest in the use of DNA polymerases (DNA pols) in next-generation sequencing strategies. These methodologies typically rely on members of the A and B family of DNA polymerases that are classified as high-fidelity DNA polymerases. These enzymes possess the ability to selectively incorporate the correct nucleotide opposite a templating base with an error frequency of only 1 in 106 insertion events. How they achieve this remarkable fidelity has been the subject of numerous investigations, yet the mechanism by which these enzymes achieve this level of accuracy remains elusive. Several smFRET assays were designed to monitor the conformational changes associated with the nucleotide selection mechanism(s) employed by DNA pols. smFRET has also been used to monitor the movement of DNA pols along a DNA substrate as well as to observe the formation of proof-reading complexes. One member among this class of enzymes, the large fragment of Bacillus stearothermophilus DNA polymerase I (Bst pol I LF), contains both 5'→3' polymerase and 3'→5' exonuclease domains, but reportedly lacks exonuclease activity. We have designed a smFRET assay showing that Bst pol I LF forms proofreading complexes. The formation of proofreading complexes at the single molecule level is strongly influenced by the presence of the 3' hydroxyl at the primer-terminus of the DNA substrate. Our assays also identify an additional state, observed in the presence of a mismatched primer-template terminus, that may be involved in the transfer of the primer-terminus from the polymerase to the exonuclease active site.
  Article Metrics


1. Joyce CM, Steitz TA (1994) Function and structure relationships in DNA polymerases. Annu Rev Biochem 63: 777–822.    

2. Joyce CM, Benkovic SJ (2004) DNA polymerase fidelity: kinetics, structure, and checkpoints. Biochemistry 43: 14317–14324.    

3. Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5: 507–516.    

4. Santoso Y, Joyce CM, Potapova O, et al. (2010) Conformational transitions in DNA polymerase I revealed by single-molecule FRET. P Natl Acad Sci USA 107: 715–720.    

5. Hohlbein J, Aigrain L, Craggs TD, et al. (2013) Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion. Nat commun 4: 2131.

6. Lamichhane R, Berezhna SY, Gill JP, et al. (2013) Dynamics of site switching in DNA polymerase. J Am Chem Soc 135: 4735–4742.    

7. Berezhna SY, Gill JP, Lamichhane R, et al. (2012) Single-molecule Forster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I. J Am Chem Soc 134: 11261–11268.    

8. Steitz TA (1999) DNA polymerases: structural diversity and common mechanisms. J biol chem 274: 17395–17398.    

9. Wu EY, Beese LS (2011) The structure of a high fidelity DNA polymerase bound to a mismatched nucleotide reveals an "ajar" intermediate conformation in the nucleotide selection mechanism. J biol chem 286: 19758–19767.    

10. Beese LS, Derbyshire V, Steitz TA (1993) Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science 260: 352–355.    

11. Beese LS, Steitz TA (1991) Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J 10: 25–33.

12. Derbyshire V, Pinsonneault JK, Joyce CM (1995) Structure-function analysis of 3'-5' exonuclease of DNA polymerases. Method Enzymol 262: 363–385.    

13. Aliotta JM, Pelletier JJ, Ware JL, et al. (1996) Thermostable Bst DNA polymerase I lacks a 3'-5' proofreading exonuclease activity. Genet Anal 12: 185–195.    

14. Rastgoo N, Sadeghizadeh M, Bambaei B, et al. (2009) Restoring 3'-5' exonuclease activity of thermophilic Geobacillus DNA polymerase I using site-directed mutagenesis in active site. J Biotechnol 144: 245–252.    

15. Kiefer JR, Mao C, Hansen CJ, et al. (1997) Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 Å resolution. Structure 5: 95–108.

16. Wang CX, Zakharova E, Li J, et al. (2004) Pre-steady-state kinetics of RB69 DNA polymerase and its exo domain mutants: effect of pH and thiophosphoryl linkages on 3'-5' exonuclease activity. Biochemistry 43: 3853–3861.    

17. Kirmizialtin S, Nguyen V, Johnson KA, et al. (2012) How conformational dynamics of DNA polymerase select correct substrates: experiments and simulations. Structure 20: 618–627.    

18. Johnson KA (2010) The kinetic and chemical mechanism of high-fidelity DNA polymerases. BBA- Proteins Proteom 1804: 1041–1048.    

19. Datta K, Johnson NP, LiCata VJ, et al. (2009) Local conformations and competitive binding affinities of single- and double-stranded primer-template DNA at the polymerization and editing active sites of DNA polymerases. J Biol Chem 284: 17180–17193.    

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

21. Previte MJ, Zhou C, Kellinger M, et al. (2015) DNA sequencing using polymerase substrate-binding kinetics. Nat Commun 6: 5936.    

22. Walsh MT, Roller EE, Ko KS, et al. (2015) Measurement of DNA polymerase incorporation kinetics of dye-labeled nucleotides using total internal reflection fluorescence microscopy. Biochemistry 54: 4019–4021.    

23. Capson TL, Peliska JA, Kaboord BF, et al. (1992) Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4. Biochemistry 31: 10984–10994.    

24. 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.    

25. Christian TD, Romano LJ, Rueda D (2009) Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution. P Natl Acad Sci USA 106: 21109–21114.    

© 2018 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution Licese (

Download full text in PDF

Export Citation

Article outline

Show full outline
Copyright © AIMS Press All Rights Reserved