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DNA binding and nucleotide flipping by the human DNA repair protein AGT

Nature Structural & Molecular Biology volume 11, pages 714–720 (2004)Cite this article

Abstract

O6-alkylguanine-DNA alkyltransferase (AGT), or O6-methylguanine-DNA methyltransferase (MGMT), prevents mutations and apoptosis resulting from alkylation damage to guanines. AGT irreversibly transfers the alkyl lesion to an active site cysteine in a stoichiometric, direct damage reversal pathway. AGT expression therefore elicits tumor resistance to alkylating chemotherapies, and AGT inhibitors are in clinical trials. We report here structures of human AGT in complex with double-stranded DNA containing the biological substrate O6-methylguanine or crosslinked to the mechanistic inhibitor N1,O6-ethanoxanthosine. The prototypical DNA major groove–binding helix-turn-helix (HTH) motif mediates unprecedented minor groove DNA binding. This binding architecture has advantages for DNA repair and nucleotide flipping, and provides a paradigm for HTH interactions in sequence-independent DNA-binding proteins like RecQ and BRCA2. Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases.

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Figure 1: Recognition and repair of O6-alkylguanines by AGT.
Figure 2: AGT-DNA binding interface.
Figure 3: Minor groove binding, nucleotide flipping and O6-akylguanine recognition by AGT.
Figure 4: Reaction mechanism.
Figure 5: Directionality of ssDNA repair by AGT.
Figure 6: The minor groove DNA-binding mechanism of AGT expands the HTH repertoire.

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Acknowledgements

We thank C.L. Brooks III, M.E. Stroupe and A.J. Das for critical discussion and the staff and facilities of the Stanford Synchrotron Radiation Laboratory. This work was supported by grants from the US National Institutes of Health (J.A.T.), US National Cancer Institute (A.E.P.), Patterson Trust (D.M.N.), Alexander and Margaret Stewart Trust (D.M.N.) and Skaggs Institute for Chemical Biology (D.S.D.).

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Author notes

  1. Tammy T Woo

    Present address: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120, Heidelberg, Germany

Authors and Affiliations

  1. Skaggs Institute for Chemical Biology, The Scripps Research Institute, MB-4, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    Douglas S Daniels, Tammy T Woo & John A Tainer

  2. Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, 17033, Pennsylvania, USA

    Kieu X Luu & Anthony E Pegg

  3. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, 21205, Maryland, USA

    David M Noll & Neil D Clarke

  4. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    John A Tainer

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Correspondence to John A Tainer.

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Daniels, D., Woo, T., Luu, K. et al. DNA binding and nucleotide flipping by the human DNA repair protein AGT. Nat Struct Mol Biol 11, 714–720 (2004). https://doi.org/10.1038/nsmb791

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