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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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.
The authors declare that they have no competing financial interests.
Primer extension assay gel showing misincorporation of dATP opposite 8oxoG and extension past A:8oxoG. (PDF 84 kb)
Stereoviews of the BF active site following dCTP or dATP incorporation opposite 8oxoG. (PDF 1013 kb)
Superposition of BF structures with A:8oxoG or a C:G base pair at the post-insertion site. (PDF 461 kb)
Plots of reaction velocity as a function of dNTP concentration used to determine kinetics for single nucleotide incorporation opposite 8oxoG or unmodified guanine. (PDF 80 kb)
Legends for Supplementary Figures 1–3 and Supplementary Data 1. (DOC 21 kb)
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Hsu, G., Ober, M., Carell, T. et al. Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase. Nature 431, 217–221 (2004). https://doi.org/10.1038/nature02908
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