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Timing facilitated site transfer of an enzyme on DNA

Nature Chemical Biology volume 8, pages 205–210 (2012)Cite this article

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Abstract

Many enzymes that react with specific sites in DNA have the property of facilitated diffusion, in which the DNA chain is used as a conduit to accelerate site location. Despite the importance of such mechanisms in gene regulation and DNA repair, there have been few viable approaches to elucidate the microscopic process of facilitated diffusion. Here we describe a new method in which a small-molecule trap (uracil) is used to clock a DNA repair enzyme as it hops and slides between damaged sites in DNA. The 'molecular clock' provides unprecedented information: the mean length for DNA sliding, the one-dimensional diffusion constant, the maximum hopping radius and the time frame for DNA hopping events. In addition, the data establish that the DNA phosphate backbone is a sufficient requirement for DNA sliding.

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Figure 1: The molecular clock approach for timing pathways for facilitated diffusion on DNA.
Figure 2: Facilitated site transfer by hUNG.
Figure 3: Pslide′ and Phop′ as functions of site spacing and strand positioning of uracils.
Figure 4: Site-transfer dependence with increasing salt and in the context of single-stranded DNA.

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Acknowledgements

We thank J. Parker (Harvard Medical School) for purified hUNG used in this study. This work was supported by US National Institutes of Health grant GM056834.

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  1. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Joseph D Schonhoft & James T Stivers

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  1. Joseph D Schonhoft
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J.D.S. performed all experiments. J.D.S. and J.T.S. analyzed the data and wrote the paper.

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Correspondence to James T Stivers.

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Schonhoft, J., Stivers, J. Timing facilitated site transfer of an enzyme on DNA. Nat Chem Biol 8, 205–210 (2012). https://doi.org/10.1038/nchembio.764

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