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Comparative Analysis of Family A DNA-Polymerases as a Searching Tool for Enzymes with New Properties

  • BIOINFORMATICS, BIOENGINEERING, AND BIOTECHNOLOGY
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Abstract—DNA polymerases catalyze DNA synthesis during DNA replication, repair, and recombination. A number of DNA polymerases, such as the Taq enzyme from Thermus aquaticus, are used in various applications of molecular biology and biotechnology, in particular as DNA amplification tools. However, the efficiency of these enzymes depends on factors such as DNA origin, primer composition, template length, GC-content, and the ability to form stable secondary structures. These limitations in the use of currently known DNA polymerases lead to the search for new enzymes with improved properties. This review summarizes the main structural and molecular-kinetic features of the functioning of DNA-polymerases belonging to structural family A, including Taq polymerase. A phylogenetic analysis of these enzymes was carried out, which made it possible to establish a highly conserved consensus sequence containing 62 amino acid residues distributed over the structure of the enzyme. A comparative analysis of these amino acid residues among poorly studied DNA-polymerases revealed 7 enzymes that potentially have the properties necessary for use in DNA amplification.

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REFERENCES

  1. Nikoomanzar A., Chim N., Yik E.J., Chaput J.C. 2020. Engineering polymerases for applications in synthetic biology. Q. Rev. Biophys. 53, 1–31.

    Article  Google Scholar 

  2. Wu D.A.N.Y., Ugozzoli L., Pal B.K., Qian J.I.N., Wallace R.B. 1991. The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. DNA Cell Biol. 10, 233–238.

    Article  CAS  PubMed  Google Scholar 

  3. Owczarzy R., Moreira B.G., You Y., Behlke M.A., Walder J.A. 2008. Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry. 47, 5336–5353.

    Article  CAS  PubMed  Google Scholar 

  4. Garcia-Diaz M., Bebenek K. 2007. Multiple functions of DNA polymerases. CRC. Crit. Rev. Plant Sci. 26, 105–122.

    Article  CAS  PubMed  Google Scholar 

  5. Alba M.M. 2001. Replicative DNA polymerases. Genome Biol. 2, 1–7.

    Article  Google Scholar 

  6. Rothwell P.J., Waksman G. 2005. Structure and mechanism of DNA polymerases. Adv. Protein Chem. 71, 401–440.

    Article  CAS  PubMed  Google Scholar 

  7. Chien A., Edgar D.B., Trela J.M. 1976. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J. Bacteriol. 127, 1550–1557.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Choi J.J., Jung S.E., Kim H.K., Kwon S.T. 1999. Purification and properties of Thermus filiformis DNA polymerase expressed in Escherichia coli. Biotechnol. Appl. Biochem. 30, 19–25.

    CAS  PubMed  Google Scholar 

  9. Lawyer F.C., Stoffel S., Saiki R.K., Chang S.Y., Landre P.A., Abramson R.D., Gelfand D.H. 1993. High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity. Genome Res. 2, 275–287.

    Article  CAS  Google Scholar 

  10. Park J.H., Kim J.S., Kwon S.-T., Lee D.-S. 1993. Purification and characterization of Thermus caldophilus GK24 DNA polymerase. Eur. J. Biochem. 214, 135–140.

    Article  CAS  PubMed  Google Scholar 

  11. Kaledin A.S., Sliusarenko A.G., Gorodetskiĭ S.I. 1980. Isolation and properties of DNA polymerase from extreme thermophylic bacteria Thermus aquaticus YT-1. Biokhimiia. 45, 644–651.

    CAS  PubMed  Google Scholar 

  12. Saiki R.K., Gelfand D.H., Stoffel S., Scharf S.J., Higuchi R., Horn G.T., Mullis K.B., Erlich H.A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 239, 487–491.

    Article  CAS  PubMed  Google Scholar 

  13. Arezi B., Xing W., Sorge J.A., Hogrefe H.H. 2003. Amplification efficiency of thermostable DNA polymerases. Anal. Biochem. 321, 226–235.

    Article  CAS  PubMed  Google Scholar 

  14. Al-Soud W.A., Rådström P. 2001. Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 39, 485–493.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Flaman J.-M., Frebourg T., Moreau V., Charbonnier F., Martin C., Ishioka C., Friend S.H., Iggo R. 1994. A rapid PCR fidelity assay. Nucleic Acids Res. 22, 3259–3260.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ling L.L., Keohavong P., Dias C., Thilly W.G. 1991. Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and Vent DNA polymerases. Genome Res. 1, 63–69.

    Article  CAS  Google Scholar 

  17. Lee J.I., Kim Y.J., Bae H., Cho S.S., Lee J.-H., Kwon S.-T. 2010. Biochemical properties and PCR performance of a family B DNA polymerase from hyperthermophilic euryarchaeon Thermococcus peptonophilus. Appl. Biochem. Biotechnol. 160, 1585–1599.

    Article  CAS  PubMed  Google Scholar 

  18. Harrell R.A., Hart R.P. 1994. Rapid preparation of Thermus flavus DNA polymerase. Genome Res. 3, 372–375.

    Article  CAS  Google Scholar 

  19. Carballeira N., Nazabal M., Brito J., Garcia O. 1990. Purification of a thermostable DNA polymerase from Thermus thermophilus HB8, useful in the polymerase chain reaction. Biotechniques. 9, 276–281.

    CAS  PubMed  Google Scholar 

  20. Yang S.-W., Astatke M., Potter J., Chatterjee D.K. 2002. Mutant Thermotoga neapolitana DNA polymerase I: altered catalytic properties for non-templated nucleotide addition and incorporation of correct nucleotides. Nucleic Acids Res. 30, 4314–4320.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Steitz T.A. 1993. DNA- and RNA-dependent DNA polymerases. Curr. Opin. Struct. Biol. 3, 31–38.

    Article  CAS  Google Scholar 

  22. Steitz T.A. 1998. A mechanism for all polymerases. Nature. 391, 231–2323.

    Article  CAS  PubMed  Google Scholar 

  23. Steitz T.A. 1999. DNA polymerases: structural diversity and common mechanisms. J. Biol. Chem. 274, 17395–17398.

    Article  CAS  PubMed  Google Scholar 

  24. Joyce C.M. 2013. DNA polymerase I, Bacterial. In Encyclopedia of Biological Chemistry, 2nd ed. Elsevier, pp. 87–90.

    Google Scholar 

  25. Betz K., Malyshev D.A., Lavergne T., Welte W., Diederichs K., Dwyer T.J., Ordoukhanian P., Romesberg F.E., Marx A. 2012. KlenTaq polymerase replicates unnatural base pairs by inducing a Watson–Crick geometry. Nat. Chem. Biol. 8, 612–614.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Raper A.T., Reed A.J., Suo Z. 2018. Kinetic mechanism of DNA polymerases: contributions of conformational dynamics and a third divalent metal ion. Chem. Rev. 118, 6000–6025.

    Article  CAS  PubMed  Google Scholar 

  27. Berdis A.J. 2009. Mechanisms of DNA polymerases. Chem. Rev. 109, 2862–2879.

    Article  CAS  PubMed  Google Scholar 

  28. Brautigam C.A., Steitz T.A. 1998. Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. Curr. Biol. 8, 54–63.

    CAS  Google Scholar 

  29. Ignatov K.B., Bashirova A.A., Miroshnikov A.I., Kramarov V.M. 1999. Mutation S543N in the thumb subdomain of the Taq DNA polymerase large fragment suppresses pausing associated with the template structure. FEBS Lett. 448, 145–148.

    Article  CAS  PubMed  Google Scholar 

  30. Drum M., Kranaster R., Ewald C., Blasczyk R., Marx A. 2014. Variants of a Thermus aquaticus DNA polymerase with increased selectivity for applications in allele- and methylation-specific amplification. PLoS One. 9, e96640.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Raghunathan G., Marx A. 2019. Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library. Sci. Rep. 9, 590.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Yamagami T., Ishino S., Kawarabayasi Y., Ishino Y. 2014. Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering. Front. Microbiol. 5. 461.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Minnick D.T., Bebenek K., Osheroff W.P., Turner R.M., Astatke M., Liu L., Kunkel T.A., Joyce C.M. 1999. Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment). J. Biol. Chem. 274, 3067–3075.

    Article  CAS  PubMed  Google Scholar 

  34. Yamagami T., Matsukawa H., Tsunekawa S., Kawarabayasi Y., Ishino S., Ishino Y. 2016. A longer finger-subdomain of family A DNA polymerases found by metagenomic analysis strengthens DNA binding and primer extension abilities. Gene. 576, 690–695.

    Article  CAS  PubMed  Google Scholar 

  35. Roberts R.J. 1995. On base flipping. Cell. 82, 9–12.

    Article  CAS  PubMed  Google Scholar 

  36. Suzuki M., Yoshida S., Adman E.T., Blank A., Loeb L.A. 2000. Thermus aquaticus DNA polymerase I mutants with altered fidelity. J. Biol. Chem. 275, 32728–32735.

    Article  CAS  PubMed  Google Scholar 

  37. Bernad A., Blanco L., Lázaro J., Martín G., Salas M. 1989. A conserved 3′→5′ exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell. 59, 219–228.

    Article  CAS  PubMed  Google Scholar 

  38. Park Y., Choi H., Lee D.S., Kim Y. 1997. Improvement of the 3′–5′ exonuclease activity of Taq DNA polymerase by protein engineering in the active site. Mol. Cells. 7, 419–424.

    CAS  PubMed  Google Scholar 

  39. Ignatov K., Kramarov V., Billingham S. 2009. Chimeric DNA polymerase. US Patent US20090209005A1.

  40. Stenesh J., McGowan G.R. 1977. DNA polymerase from mesophilic and thermophilic bacteria. Biochim. Biophys. Acta, Nucleic Acids Protein Synth. 475, 32–41.

    Article  CAS  Google Scholar 

  41. Kevbrin V.V., Zengler K., Lysenko A., Wiegel J. 2005. Anoxybacillus kamchatkensis sp. nov., a novel thermophilic facultative aerobic bacterium with a broad pH optimum from the Geyser valley, Kamchatka. Extremophiles. 9, 391–398.

    Article  CAS  PubMed  Google Scholar 

  42. Namsaraev Z., Babasanova O., Dunaevsky Y., Akimov V., Barkhutova D., Gorlenko V.M., Namsaraev B. 2010. Anoxybacillus mongoliensis sp. nov., a novel thermophilic proteinase producing bacterium isolated from alkaline hot spring, central Mongolia. Microbiology (Moscow). 79, 491‒499. https://doi.org/10.1134/S0026261710040119

    Article  CAS  Google Scholar 

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Funding

The work was supported by the Ministry of Science and Higher Education, agreement no. 075-15-2021-1085.

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A. A. Bulygin and A. A. Kuznetsova contributed equally to this review.

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Correspondence to N. A. Kuznetsov.

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Bulygin, A.A., Kuznetsova, A.A., Fedorova, O.S. et al. Comparative Analysis of Family A DNA-Polymerases as a Searching Tool for Enzymes with New Properties. Mol Biol 57, 182–192 (2023). https://doi.org/10.1134/S0026893323020048

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