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Genome instability due to ribonucleotide incorporation into DNA

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

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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.

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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.

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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.).

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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

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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.

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Correspondence to Thomas A Kunkel.

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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

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