Abstract
DNA polymerases recognize their substrates with exceptionally high specificity1,2, restricting the use of unnatural nucleotides and the applications they enable. We describe a strategy to expand the substrate range of polymerases. By selecting for the extension of distorting 3′ mismatches, we obtained mutants of Taq DNA polymerase that not only promiscuously extended mismatches, but had acquired a generic ability to process a diverse range of noncanonical substrates while maintaining high catalytic turnover, processivity and fidelity. Unlike the wild-type enzyme, they bypassed blocking lesions such as an abasic site, a thymidine dimer or the base analog 5-nitroindol3 and performed PCR amplification with complete substitution of all four nucleotide triphosphates with phosphorothioates4 or the substitution of one with the equivalent fluorescent dye–labeled nucleotide triphosphate. Such 'unfussy' polymerases have immediate utility, as we demonstrate by the generation of microarray probes with up to 20-fold brighter fluorescence.
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References
Kunkel, T.A. & Bebenek, K. DNA replication fidelity. Annu. Rev. Biochem. 69, 497–529 (2000).
Kool, E.T. Active site tightness and substrate fit in DNA replication. Annu. Rev. Biochem. 71, 191–219 (2002).
Loakes, D. Survey and summary: The applications of universal DNA base analogues. Nucleic Acids Res. 29, 2437–2447 (2001).
Verma, S. & Eckstein, F. Modified oligonucleotides: synthesis and strategy for users. Annu. Rev. Biochem. 67, 99–134 (1998).
Li, Y., Mitaxov, V. & Waksman, G. Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation. Proc. Natl. Acad. Sci. USA 96, 9491–9496 (1999).
Astatke, M., Ng, K., Grindley, N.D. & Joyce, C.M. A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides. Proc. Natl. Acad. Sci. USA 95, 3402–3407 (1998).
Xia, G. et al. Directed evolution of novel polymerase activities: mutation of a DNA polymerase into an efficient RNA polymerase. Proc. Natl. Acad. Sci. USA 99, 6597–6602 (2002).
Jestin, J.L., Kristensen, P. & Winter, G. A method for the selection of catalytic activity using phage display and proximity coupling. Angew. Chem. Int. Ed. 38, 1124–1127 (1999).
Huang, M.-M., Arnheim, N. & Goodman, M.F. Extension of base mispairs by Taq polymerase: implications for single nucleotide discrimination in PCR. Nucleic Acids Res. 20, 4567–4573 (1992).
Ghadessy, F.J., Ong, J.L. & Holliger, P. Directed evolution of polymerase function by compartmentalized self-replication. Proc. Natl. Acad. Sci. USA 98, 4552–4557 (2001).
Kwok, S. et al. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 18, 999–1005 (1990).
Goodman, M.F. Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu. Rev. Biochem. 71, 17–50 (2002).
Friedberg, E.C., Wagner, R. & Radman, M. Specialized DNA polymerases, cellular survival, and the genesis of mutations. Science 296, 1627–1630 (2002).
Barnes, W.M. PCR amplification of up to 35-kb DNA with high-fidelity and high-yield from l bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216–2220 (1994).
Ling, H., Boudsocq, F., Woodgate, R. & Yang, W. Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication. Cell 107, 91–102 (2001).
Trincao, J. et al. Structure of the catalytic core of S. cerevisiae DNA polymerase eta: implications for translesion DNA synthesis. Mol. Cell 8, 417–426 (2001).
Kool, E.T. Synthetically modified DNAs as substrates for polymerases. Curr. Opin. Chem. Biol. 4, 602–608 (2000).
Morales, J.C. & Kool, E.T. Functional hydrogen-bonding map of the minor groove binding tracks of six DNA polymerases. Biochemistry 39, 12979–12988 (2000).
Tae, E.L., Wu, Y., Xia, G., Schultz, P.G. & Romesberg, F.E. Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs. J. Am. Chem. Soc. 123, 7439–7440 (2001).
Piccirilli, J.A., Krauch, T., Moroney, S.E. & Benner, S.A. Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature 343, 33–37 (1990).
Engelke, D.R., Krikos, A., Bruck, M.E. & Ginsburg, D. Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Anal. Biochem. 191, 396–400 (1990).
Zhao, H., Giver, L., Shao, Z., Affholter, J.A. & Arnold, F.H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16, 258–261 (1998).
Creighton, S., Bloom, L.B. & Goodman, M.F. Gel fidelity assay measuring nucleotide misinsertion, exonucleolytic proofreading, and lesion bypass efficiencies. Methods Enzymol. 262, 232–256 (1995).
Murata, T., Iwai, S. & Ohtsuka, E. Synthesis and characterization of a substrate for T4 endonuclease V containing a phosphorodithioate linkage at the thymine dimer site. Nucleic Acids Res. 18, 7279–7286 (1990).
Kokoska, R.J., McCulloch, S.D. & Kunkel, T.A. The efficiency and specificity of apurinic/apyrimidinic site bypass by human DNA polymerase eta and Sulfolobus solfataricus Dpo4. J. Biol. Chem. 278, 50537–50545 (2003).
Debbie, P. et al. Allele identification using immobilized mismatch binding protein: Detection and identification of antibiotic resistant bacteria and determination of sheep susceptibility to scrapie. Nucleic Acids Res. 25, 4825–4829 (1997).
Eom, S.H., Wang, J. & Steitz, T.A. Structure of Taq polymerase with DNA at the polymerase active site. Nature 382, 278–281 (1996).
Acknowledgements
F.B., A.V. and R.W. were supported by funds from the National Institutes of Health intramural research program.
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Ghadessy, F., Ramsay, N., Boudsocq, F. et al. Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution. Nat Biotechnol 22, 755–759 (2004). https://doi.org/10.1038/nbt974
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DOI: https://doi.org/10.1038/nbt974
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