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Dynamic opening of DNA during the enzymatic search for a damaged base

Nature Structural & Molecular Biology volume 11, pages 1230–1236 (2004)Cite this article

Abstract

Uracil DNA glycosylase (UDG) removes uracil from U·A or U·G base pairs in genomic DNA by extruding the aberrant uracil from the DNA base stack. A question in enzymatic DNA repair is whether UDG and related glycosylases also use an extrahelical recognition mechanism to inspect the integrity of undamaged base pairs. Using NMR imino proton exchange measurements we find that UDG substantially increases the equilibrium constant for opening of T-A base pairs by almost two orders of magnitude relative to free B-DNA. This increase is brought about by enzymatic stabilization of an open state of the base pair without increasing the rate constant for spontaneous base pair opening. These findings indicate a passive search mechanism in which UDG uses the spontaneous opening dynamics of DNA to inspect normal base pairs in a rapid genome-wide search for uracil in DNA.

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Figure 1: Active and two passive mechanisms for base flipping.
Figure 2: Imino proton spectra and sequences of DNA duplexes used in these studies (pH 8.0, 10 °C).
Figure 3: Water magnetization transfer kinetics at pH 8.0 and 10 °C in the absence of DFEA buffer catalyst (see Supplementary Methods online).
Figure 4: Imino proton exchange mechanism and double-reciprocal plots of the observed exchange rates against DFEA catalyst concentration (see equation (1)).
Figure 5: Relative imino proton exchange parameters for the free and UDG-bound T-A duplex.

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Acknowledgements

This work was supported by US National Institutes of Health grant GM56834-09 to J.T.S.

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Authors and Affiliations

  1. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, 21205, Maryland, USA

    Chunyang Cao, Yu Lin Jiang & James T Stivers

  2. Center for Advanced Research in Biotechnology of the National Institute of Standards and Technology and the University of Maryland Biotechnology Institutes, 9600 Gudelsky Drive, Rockville, 20850, Maryland, USA

    Fenhong Song

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Supplementary information

Supplementary Fig. 1

Determination of the equilibrium dissociation constants for the T-A and G-C duplexes. (PDF 52 kb)

Supplementary Table 1

Rate and equilibrium constants for base pair opening in free and bound T/A duplex DNA. (PDF 43 kb)

Supplementary Table 2

Rate and equilibrium constants for base pair opening in 2′-FU·A duplex DNA. (PDF 34 kb)

Supplementary Table 3

Rate and equilibrium constants for base pair opening in free and bound G-C duplex DNA. (PDF 36 kb)

Supplementary Methods (PDF 58 kb)

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Cao, C., Jiang, Y., Stivers, J. et al. Dynamic opening of DNA during the enzymatic search for a damaged base. Nat Struct Mol Biol 11, 1230–1236 (2004). https://doi.org/10.1038/nsmb864

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