Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

An orthogonal DNA replication system in yeast

Abstract

An extranuclear replication system, consisting of an orthogonal DNA plasmid–DNA polymerase pair, was developed in Saccharomyces cerevisiae. Engineered error-prone DNA polymerases showed complete mutational targeting in vivo: per-base mutation rates on the plasmid were increased substantially and remained stable with no increase in genomic rates. Orthogonal replication serves as a platform for in vivo continuous evolution and as a system whose replicative properties can be manipulated independently of the host's.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: An orthogonal replication system based on the p1/2 replication system.

References

  1. Drake, J.W. Proc. Natl. Acad. Sci. USA 88, 7160–7164 (1991).

    CAS  Article  Google Scholar 

  2. Nowak, M.A. Trends Ecol. Evol. 7, 118–121 (1992).

    CAS  Article  Google Scholar 

  3. Camps, M., Naukkarinen, J., Johnson, B.P. & Loeb, L.A. Proc. Natl. Acad. Sci. USA 100, 9727–9732 (2003).

    CAS  Article  Google Scholar 

  4. Esvelt, K.M., Carlson, J.C. & Liu, D.R. Nature 472, 499–503 (2011).

    CAS  Article  Google Scholar 

  5. Klassen, R. & Meinhardt, R. Microbiol. Monogr. 7, 187–226 (2007).

    Article  Google Scholar 

  6. Gunge, N. & Sakaguchi, K. J. Bacteriol. 147, 155–160 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Kämper, J., Esser, K., Gunge, N. & Meinhardt, F. Curr. Genet. 19, 109–118 (1991).

    Article  Google Scholar 

  8. Foster, P.L. Methods Enzymol. 409, 195–213 (2006).

    CAS  Article  Google Scholar 

  9. Ma, W.T., Sandri, G.v.H. & Sarkar, S. J. Appl. Prob. 29, 255–267 (1992).

    Article  Google Scholar 

  10. Hall, B.M., Ma, C., Liang, P. & Singh, K.K. Bioinformatics 25, 1564–1565 (2009).

    CAS  Article  Google Scholar 

  11. Lang, G.I. & Murray, A.W. Genetics 178, 67–82 (2008).

    CAS  Article  Google Scholar 

  12. Jung, G.H., Leavitt, M.C. & Ito, J. Nucleic Acids Res. 15, 9088 (1987).

    CAS  Article  Google Scholar 

  13. Uil, T.G. et al. Nucleic Acids Res. 39, e30 (2011).

    CAS  Article  Google Scholar 

  14. Finney-Manchester, S.P. & Maheshri, N. Nucleic Acids Res. 41, e99 (2013).

    CAS  Article  Google Scholar 

  15. Rose, M.D. Annu. Rev. Microbiol. 45, 539–567 (1991).

    CAS  Article  Google Scholar 

  16. Liu, C.C. & Schultz, P.G. Annu. Rev. Biochem. 79, 413–444 (2010).

    CAS  Article  Google Scholar 

  17. Rackham, O. & Chin, J.W. Nat. Chem. Biol. 1, 159–166 (2005).

    CAS  Article  Google Scholar 

  18. An, W. & Chin, J.W. Proc. Natl. Acad. Sci. USA 106, 8477–8482 (2009).

    CAS  Article  Google Scholar 

  19. Gibson, D.G. et al. Nat. Methods 6, 343–345 (2009).

    CAS  Article  Google Scholar 

  20. Burker, D.J., Amberg, D.C. & Strathern, J.N. Methods in Yeast Genetics: a Cold Spring Harbor Laboratory Course Manual (CSHL Press, New York, 2005).

  21. Frishman, F. in A Modern Course on Statistical Distributions in Scientific Work (eds. Patil, G.P., Kotz, S. & Ord, J.K.) 401–406 (Proceedings of the NATO Advanced Study Institute, 1975).

  22. Smiley, J.A. & Jones, M.E. Biochemistry 31, 12162–12168 (1992).

    CAS  Article  Google Scholar 

  23. Miller, B.G. & Wolfenden, R. Annu. Rev. Biochem. 71, 847–885 (2002).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This research was funded by generous startup funds from the University of California–Irvine to C.C.L. and a US National Science Foundation Graduate Research Fellowship to A.R. We are greatly indebted to A.P. Arkin and members of the Miller Institute of the University of California–Berkeley for early encouragement of this idea. We thank R. Rezvani, K.F. Kearns and K.H. Spencer for experimental assistance. We thank J.E. Dueber (University of California–Berkeley) for helpful discussions and the gift of certain yeast expression plasmids. We thank P.G. Schultz, J.A. Wells and G.A. Weiss for valuable comments on our work.

Author information

Authors and Affiliations

Authors

Contributions

C.C.L. conceived this project; A.R. and C.C.L. designed all of the experiments; A.R., A.A. and C.C.L. performed all of the experiments; and A.R. and C.C.L. analyzed all of the data and wrote the paper.

Corresponding author

Correspondence to Chang C Liu.

Ethics declarations

Competing interests

A provisional patent on this work has been filed.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–7 and Supplementary Tables 1–6. (PDF 2105 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ravikumar, A., Arrieta, A. & Liu, C. An orthogonal DNA replication system in yeast. Nat Chem Biol 10, 175–177 (2014). https://doi.org/10.1038/nchembio.1439

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1439

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing