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Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

Nature Chemical Biology volume 4, pages 51–58 (2008)Cite this article

Abstract

Escherichia coli MutY has an important role in preventing mutations associated with the oxidative lesion 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG) in DNA by excising adenines from OG·A mismatches as the first step of base excision repair. To determine the importance of specific steps in the base pair recognition and base removal process of MutY, we have evaluated the effects of modifications of the OG·A substrate on the kinetics of base removal, mismatch affinity and repair to G-C in an E. coli–based assay. Notably, adenine modification was tolerated in the cellular assay, whereas modification of OG resulted in minimal cellular repair. High affinity for the mismatch and efficient base removal required the presence of OG. Taken together, these results suggest that the presence of OG is a critical feature that is necessary for MutY to locate OG·A mismatches and select the appropriate adenines for excision to initiate repair in vivo before replication.

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Figure 1: Recognition of an OG·A mismatch by D144N BsMY and modifications made to probe features of mismatch repair.
Figure 2: Kinetic analysis of the glycosylase activity of MutY.
Figure 3: Cell-based MutY-mediated repair assay.

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Acknowledgements

We thank B. Wilcock, S. Kundu and M.A. Pope for technical assistance. We also thank M. Marinus (University of Massachusetts) for providing the AB1157, GM7724 (mutY::Cam) and KM75 (mutS465::Tet) E. coli strains. J. Miller and M. Michaels (University of California, Los Angeles) provided the pKKYEco plasmid and JM101 (mutY−) E. coli strain. This work was supported by US National Institutes of Health (NIH) grants to S.S.D. (CA67985) and E.T.K. (GM072705), and by NIH predoctoral traineeships to A.L.L. (GM08537) and V.L.O. (GM08537 and CA093247). The DNA sequencing facility at the University of Utah Medical School is supported in part by an NIH National Cancer Institute grant (5P30CA43014).

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Author notes

  1. Alison L Livingston and Valerie L O'Shea: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, 84112, Utah, USA

    Alison L Livingston & Valerie L O'Shea

  2. Department of Chemistry, Stanford University, Stauffer 1, Stanford, 94305, California, USA

    Taewoo Kim & Eric T Kool

  3. Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, 95616, California, USA

    Sheila S David

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  1. Alison L Livingston
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Contributions

A.L.L., V.L.O. and S.S.D. designed the experiments. A.L.L. and V.L.O. performed the experiments. T.K. synthesized the Z3-containing duplex 2 oligonucleotide. S.S.D. and E.T.K. supervised all experiments. A.L.L., V.L.O. and S.S.D. wrote the manuscript. E.T.K. read and made comments on the manuscript.

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Correspondence to Sheila S David.

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Livingston, A., O'Shea, V., Kim, T. et al. Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY. Nat Chem Biol 4, 51–58 (2008). https://doi.org/10.1038/nchembio.2007.40

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