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Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase

Nature volume 431, pages 217221 (09 September 2004) | Download Citation

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Abstract

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome1. Oxidative damage is implicated in ageing2 and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine3. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers4,5. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG·adenine mismatch mimics a cognate base pair whereas the 8oxoG·cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.

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Acknowledgements

We thank H. W. Hellinga for critical reading of the manuscript. Research was carried out in part at the SER-CAT 22-ID beamline at the Advanced Photon Source (Argonne National Laboratory), which is supported by the US Department of Energy, Office of Energy Research. We also thank J. J. Warren for assistance with data collection. The work was supported by grants to L.S.B. from the National Cancer Institute and the Duke Comprehensive Cancer Center, and to T.C. from the Deutsche Forschungsgemeinschaft, the Volkswagen Stiftung and the Fonds of the German Chemical Industry. M.O. is supported by a fellowship from the Boehringer Ingelheim Foundation.

Author information

Affiliations

  1. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA

    • Gerald W. Hsu
    •  & Lorena S. Beese
  2. Department of Chemistry and Biochemistry, Ludwig Maximilians University Munich, Butenandtstrasse 5-13, D 81377 Munich, Germany

    • Matthias Ober
    •  & Thomas Carell

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Competing interests

The authors declare that they have no competing financial interests.

Corresponding author

Correspondence to Lorena S. Beese.

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    Primer extension assay gel showing misincorporation of dATP opposite 8oxoG and extension past A:8oxoG.

  2. 2.

    Supplementary Figure 2

    Stereoviews of the BF active site following dCTP or dATP incorporation opposite 8oxoG.

  3. 3.

    Supplementary Figure 3

    Superposition of BF structures with A:8oxoG or a C:G base pair at the post-insertion site.

  4. 4.

    Supplementary Data 1

    Plots of reaction velocity as a function of dNTP concentration used to determine kinetics for single nucleotide incorporation opposite 8oxoG or unmodified guanine.

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    Supplementary Figure Legends

    Legends for Supplementary Figures 1–3 and Supplementary Data 1.

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DOI

https://doi.org/10.1038/nature02908

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