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Article
Nature Structural & Molecular Biology  11, 714 - 720 (2004)
Published online: 27 June 2004; | doi:10.1038/nsmb791

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

Douglas S Daniels1, Tammy T Woo1, 5, Kieu X Luu2, David M Noll3, Neil D Clarke3, Anthony E Pegg2 & John A Tainer1, 4

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

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

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

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

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

Correspondence should be addressed to John A Tainer jat@scripps.edu
O 6-alkylguanine-DNA alkyltransferase (AGT), or O 6-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 O 6-methylguanine or crosslinked to the mechanistic inhibitor N 1,O 6-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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Nature Structural & Molecular Biology
ISSN: 1545-9993
EISSN: 1545-9985
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