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Uracil-DNA glycosylase acts by substrate autocatalysis

Nature volume 413, pages 752–755 (2001)Cite this article

Abstract

In humans, uracil appears in DNA at the rate of several hundred bases per cell each day as a result of misincorporation of deoxyuridine (dU) or deamination of cytosine. Four enzymes that catalyse the hydrolysis of the glycosylic bond of dU in DNA to yield an apyridiminic site as the first step in base excision repair have been identified in the human genome1. The most efficient and well characterized of these uracil-DNA glycosylases is UDG (also known as UNG and present in almost all known organisms)2, which excises U from single- or double-stranded DNA and is associated with DNA replication forks3. We used a hybrid quantum-mechanical/molecular-mechanical (QM/MM) approach4 to determine the mechanism of catalysis by UDG. In contrast to the concerted associative mechanism proposed initially5,6,7,8,9,10, we show here that the reaction proceeds in a stepwise dissociative manner11,12. Cleavage of the glycosylic bond yields an intermediate comprising an oxocarbenium cation and a uracilate anion. Subsequent attack by a water molecule and transfer of a proton to D145 result in the products. Surprisingly, the primary contribution to lowering the activation energy comes from the substrate, rather than from the enzyme. This ‘autocatalysis’ derives from the burial and positioning of four phosphate groups that stabilize the rate-determining transition state. The importance of these phosphates explains the residual activity observed for mutants that lack key residues6,7,8,9. A corresponding catalytic mechanism could apply to the DNA glycosylases TDG and SMUG1, which belong to the same structural superfamily as UDG13,14.

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Figure 1: Reaction mechanism.
Figure 2: Wall-eyed stereodiagram of TS1.
Figure 3: Variation of the TS1 barrier height relative to reactants as MM charges are set to zero cumulatively in order of decreasing distance from C1′ of dU in TS1.

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Acknowledgements

We thank N. H. Williams for a critical reading of the manuscript; X. Lopez, Q. Cui and R. Petrella for helpful discussions; B. Webb for technical assistance; and W. G. Richards for his hospitality in Oxford, where most of the work was carried out. A.R.D. is a Burroughs Wellcome Fund Hitchings-Elion Postdoctoral Fellow, and, when the research was initiated, M.K. was the Eastman Visiting Professor in Oxford. The work at Sheffield is supported by a grant from the Biotechnology and Biological Science Research Council; the work at Harvard is supported in part by a grant from the National Institutes of Health.

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  1. Aaron R. Dinner

    Present address: Department of Chemistry, University of California, Berkeley, California, 94720, USA

Authors and Affiliations

  1. Central Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QH, UK

    Aaron R. Dinner & Martin Karplus

  2. Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield, S3 7HF, UK

    G. Michael Blackburn

  3. Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, 02138, Massachusetts, USA

    Martin Karplus

  4. Laboratoire de Chimie Biophysique, ISIS, Université Louis Pasteur, 4 Rue Blaise Pascal, Strasbourg, 67000, France

    Martin Karplus

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Correspondence to Martin Karplus.

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Dinner, A., Blackburn, G. & Karplus, M. Uracil-DNA glycosylase acts by substrate autocatalysis. Nature 413, 752–755 (2001). https://doi.org/10.1038/35099587

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