Letter | Published:

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

Nature volume 517, pages 635639 (29 January 2015) | Download Citation

Abstract

Oxidative stress promotes genomic instability and human diseases1. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2′-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP)2,3. Nucleotide pools are especially vulnerable to oxidative damage4. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival5,6 and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner7. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics8. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis9,10. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine11. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

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Accessions

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession numbers 4UAW, 4UAY, 4UAZ, 4UB1, 4UB2, 4UB3, 4UB4, 4UB5, 4UBB, and 4UBC.

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Acknowledgements

We thank the Collaborative Crystallography group at the National Institute of Environmental Health Sciences for help with data collection and analysis. We thank L. Pedersen for discussions. Use of the advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract W-31-109-Eng-38. This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (project numbers Z01-ES050158 (to S.W.), Z01-ES050161 (to S.W.), and ZIC-ES043010 (to L.P.)) and in association with National Institutes of Health grant 1U19CA105010. We are grateful for computational support for the molecular dynamics simulations from the HPC clusters at NYU as well as the Blue Gene at CCNI. Support from Philip Morris USA Inc. and Philip Morris International to T.S. is gratefully acknowledged.

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Affiliations

  1. Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, PO Box 12233, Research Triangle Park, North Carolina 27709-2233, USA

    • Bret D. Freudenthal
    • , William A. Beard
    • , Lalith Perera
    • , David D. Shock
    •  & Samuel H. Wilson
  2. Department of Chemistry, New York University, and NYU-ECNU Center for Computational Chemistry at NYU Shanghai, 10th Floor Silver Center, 100 Washington Square East, New York, New York 10003, USA

    • Taejin Kim
    •  & Tamar Schlick
  3. Courant Institute of Mathematical Sciences, New York University, 251 Mercer Street, New York, New York 10012, USA

    • Taejin Kim
    •  & Tamar Schlick

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Contributions

B.F., W.B., and S.W. designed the project. B.F. performed crystallography. D.S. did the kinetic analyses. T.K. and T.S. did the molecular dynamics simulations. L.P. did the quantum mechanical analysis. B.F., W.B., and S.W. prepared the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Samuel H. Wilson.

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https://doi.org/10.1038/nature13886

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