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.

  • Article
  • Published:

Genome instability due to ribonucleotide incorporation into DNA

Nature Chemical Biology volume 6, pages 774–781 (2010)Cite this article

Subjects

Abstract

Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine whether ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active-site methionine in yeast DNA polymerase ϵ (Pol ϵ). Ribonucleotide incorporation in vitro was three-fold lower for M644L and 11-fold higher for M644G Pol ϵ compared to wild-type Pol ϵ. This hierarchy was recapitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Δ strain progressed more slowly through S phase, had elevated dNTP pools and generated 2–5-base-pair deletions in repetitive sequences at a high rate and in a gene orientation–dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2–dependent repair and that defective repair results in replicative stress and genome instability via DNA strand misalignment.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: rNMP incorporation and bypass by Pol ϵ derivatives.
Figure 2: rNMPs in genomic DNA of Pol ϵ strains ± RNase H2.
Figure 3: Characteristics of Pol ϵ strains ± RNase H2.
Figure 4: Mutational spectra in pol2-M644G rnh201Δ strains.

Similar content being viewed by others

References

  1. Joyce, C.M. Choosing the right sugar: how polymerases select a nucleotide substrate. Proc. Natl. Acad. Sci. USA 94, 1619–1622 (1997).

    Article  CAS  Google Scholar 

  2. Nick McElhinny, S.A. et al. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proc. Natl. Acad. Sci. USA 107, 4949–4954 (2010).

    Article  CAS  Google Scholar 

  3. Kornberg, A. & Baker, T.A. DNA Replication 2nd ed. (W.H. Freeman, New York, 1992).

  4. Traut, T.W. Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 1–22 (1994).

    Article  CAS  Google Scholar 

  5. Pursell, Z.F., Isoz, I., Lundstrom, E.B., Johansson, E. & Kunkel, T.A. Yeast DNA polymerase epsilon participates in leading-strand DNA replication. Science 317, 127–130 (2007).

    Article  CAS  Google Scholar 

  6. Nick McElhinny, S.A., Gordenin, D.A., Stith, C.M., Burgers, P.M. & Kunkel, T.A. Division of labor at the eukaryotic replication fork. Mol. Cell 30, 137–144 (2008).

    Article  CAS  Google Scholar 

  7. Rossi, M.L., Purohit, V., Brandt, P.D. & Bambara, R.A. Lagging strand replication proteins in genome stability and DNA repair. Chem. Rev. 106, 453–473 (2006).

    Article  CAS  Google Scholar 

  8. Burgers, P.M. Polymerase dynamics at the eukaryotic DNA replication fork. J. Biol. Chem. 284, 4041–4045 (2009).

    Article  CAS  Google Scholar 

  9. DeLucia, A.M., Grindley, N.D. & Joyce, C.M. An error-prone family Y DNA polymerase (DinB homolog from Sulfolobus solfataricus) uses a 'steric gate' residue for discrimination against ribonucleotides. Nucleic Acids Res. 31, 4129–4137 (2003).

    Article  CAS  Google Scholar 

  10. Bonnin, A., Lazaro, J.M., Blanco, L. & Salas, M. A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase. J. Mol. Biol. 290, 241–251 (1999).

    Article  CAS  Google Scholar 

  11. Gardner, A.F. & Jack, W.E. Determinants of nucleotide sugar recognition in an archaeon DNA polymerase. Nucleic Acids Res. 27, 2545–2553 (1999).

    Article  CAS  Google Scholar 

  12. Yang, G., Franklin, M., Li, J., Lin, T.C. & Konigsberg, W. A conserved Tyr residue is required for sugar selectivity in a Pol alpha DNA polymerase. Biochemistry 41, 10256–10261 (2002).

    Article  CAS  Google Scholar 

  13. Pavlov, Y.I., Shcherbakova, P.V. & Kunkel, T.A. In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta. Genetics 159, 47–64 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 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).

    Article  CAS  Google Scholar 

  15. Shinkai, A., Patel, P.H. & Loeb, L.A. The conserved active site motif A of Escherichia coli DNA polymerase I is highly mutable. J. Biol. Chem. 276, 18836–18842 (2001).

    Article  CAS  Google Scholar 

  16. Rydberg, B. & Game, J. Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts. Proc. Natl. Acad. Sci. USA 99, 16654–16659 (2002).

    Article  CAS  Google Scholar 

  17. Eder, P.S. & Walder, J.A. Ribonuclease H from K562 human erythroleukemia cells. Purification, characterization, and substrate specificity. J. Biol. Chem. 266, 6472–6479 (1991).

    CAS  PubMed  Google Scholar 

  18. Eder, P.S., Walder, R.Y. & Walder, J.A. Substrate specificity of human RNase H1 and its role in excision repair of ribose residues misincorporated in DNA. Biochimie 75, 123–126 (1993).

    Article  CAS  Google Scholar 

  19. Cerritelli, S.M. & Crouch, R.J. Ribonuclease H: the enzymes in eukaryotes. FEBS J. 276, 1494–1505 (2009).

    Article  CAS  Google Scholar 

  20. Pursell, Z.F., Isoz, I., Lundstrom, E.B., Johansson, E. & Kunkel, T.A. Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase epsilon. Nucleic Acids Res. 35, 3076–3086 (2007).

    Article  CAS  Google Scholar 

  21. Chabes, A. et al. Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase. Cell 112, 391–401 (2003).

    Article  CAS  Google Scholar 

  22. Qiu, J., Qian, Y., Frank, P., Wintersberger, U. & Shen, B. Saccharomyces cerevisiae RNase H(35) functions in RNA primer removal during lagging-strand DNA synthesis, most efficiently in cooperation with Rad27 nuclease. Mol. Cell. Biol. 19, 8361–8371 (1999).

    Article  CAS  Google Scholar 

  23. Chen, J.Z., Qiu, J., Shen, B. & Holmquist, G.P. Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR. Nucleic Acids Res. 28, 3649–3656 (2000).

    Article  CAS  Google Scholar 

  24. Jeong, H.S., Backlund, P.S., Chen, H.C., Karavanov, A.A. & Crouch, R.J. RNase H2 of Saccharomyces cerevisiae is a complex of three proteins. Nucleic Acids Res. 32, 407–414 (2004).

    Article  CAS  Google Scholar 

  25. Frank, P., Braunshofer-Reiter, C., Karwan, A., Grimm, R. & Wintersberger, U. Purification of Saccharomyces cerevisiae RNase H(70) and identification of the corresponding gene. FEBS Lett. 450, 251–256 (1999).

    Article  CAS  Google Scholar 

  26. Bebenek, K. & Kunkel, T.A. Frameshift errors initiated by nucleotide misincorporation. Proc. Natl. Acad. Sci. USA 87, 4946–4950 (1990).

    Article  CAS  Google Scholar 

  27. Kunkel, T.A., Hamatake, R.K., Motto-Fox, J., Fitzgerald, M.P. & Sugino, A. Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae. Mol. Cell. Biol. 9, 4447–4458 (1989).

    Article  CAS  Google Scholar 

  28. Cai, H., Yu, H., McEntee, K., Kunkel, T.A. & Goodman, M.F. Purification and properties of wild-type and exonuclease-deficient DNA polymerase II from Escherichia coli. J. Biol. Chem. 270, 15327–15335 (1995).

    Article  CAS  Google Scholar 

  29. Crow, Y.J. et al. Mutations in genes encoding ribonuclease H2 subunits cause Aicardi-Goutieres syndrome and mimic congenital viral brain infection. Nat. Genet. 38, 910–916 (2006).

    Article  CAS  Google Scholar 

  30. Hamatake, R.K. et al. Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae. Identification of the catalytic core and a possible holoenzyme form of the enzyme. J. Biol. Chem. 265, 4072–4083 (1990).

    CAS  PubMed  Google Scholar 

  31. Chilkova, O., Jonsson, B.H. & Johansson, E. The quaternary structure of DNA polymerase epsilon from Saccharomyces cerevisiae. J. Biol. Chem. 278, 14082–14086 (2003).

    Article  CAS  Google Scholar 

  32. Kokoska, R.J., McCulloch, S.D. & Kunkel, T.A. The efficiency and specificity of apurinic/apyrimidinic site bypass by human DNA polymerase η and Sulfolobus solfataricus Dpo4. J. Biol. Chem. 278, 50537–50545 (2003).

    Article  CAS  Google Scholar 

  33. Pavlov, Y.I. et al. Correlation of somatic hypermutation specificity and A-T base pair substitution errors by DNA polymerase eta during copying of a mouse immunoglobulin kappa light chain transgene. Proc. Natl. Acad. Sci. USA 99, 9954–9959 (2002).

    Article  CAS  Google Scholar 

  34. Kirchner, J.M., Tran, H. & Resnick, M.A.A. DNA polymerase epsilon mutant that specifically causes +1 frameshift mutations within homonucleotide runs in yeast. Genetics 155, 1623–1632 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Journal, 2nd Edition (Cold Spring Harbor Press, Cold Spring Harbor, New York), 6.20–6.21 (1989).

  36. Sabouri, N., Viberg, J., Goyal, D.K., Johansson, E. & Chabes, A. Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage. Nucleic Acids Res. 36, 5660–5667 (2008).

    Article  CAS  Google Scholar 

  37. Shcherbakova, P.V. & Kunkel, T.A. Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations. Mol. Cell. Biol. 19, 3177–3183 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Bebenek and J. Williams for thoughtful comments on the manuscript and the National Institute of Environmental Health Sciences Molecular Genetics Core for technical support in DNA sequence analysis of ura3 mutants. This work was supported in part by Project Z01 ES065070 (to T.A.K.) from the Division of Intramural Research of the US National Institutes of Health, National Institute of Environmental Health Sciences, in part by the Swedish Foundation for Strategic Research, the Swedish Research Council and the Swedish Cancer Society (to A.C.) and in part by the Swedish Research Council, the Swedish Cancer Society, Smärtafonden and the fund for Basic Science–oriented Biotechnology, Medical Faculty of Umeå University (to E.J.).

Author information

Authors and Affiliations

  1. Department of Health and Human Services, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Stephanie A Nick McElhinny, Alan B Clark, Danielle L Watt, Brian E Watts & Thomas A Kunkel

  2. Department of Health and Human Services, Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Stephanie A Nick McElhinny, Alan B Clark, Danielle L Watt, Brian E Watts & Thomas A Kunkel

  3. Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden

    Dinesh Kumar, Else-Britt Lundström, Erik Johansson & Andrei Chabes

  4. Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden

    Andrei Chabes

Authors
  1. Stephanie A Nick McElhinny
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dinesh Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alan B Clark
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Danielle L Watt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Brian E Watts
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Else-Britt Lundström
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Erik Johansson
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Andrei Chabes
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Thomas A Kunkel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.A.N.M., D.K., A.B.C., B.E.W. and D.L.W. performed the experiments and analyzed the data; E.-B.L. contributed reagents; E.J., A.C. and T.A.K. designed the experiments and analyzed data; T.A.K. wrote the manuscript; and all authors edited the manuscript.

Corresponding author

Correspondence to Thomas A Kunkel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

McElhinny, S., Kumar, D., Clark, A. et al. Genome instability due to ribonucleotide incorporation into DNA. Nat Chem Biol 6, 774–781 (2010). https://doi.org/10.1038/nchembio.424

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.424

This article is cited by

Search

Advanced 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