ARTICLES 714 VOLUME 11 NUMBER 8 AUGUST 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY DNA repair proteins carry out the essential function of protecting the genomic integrity of cells from both endogenous and exogenous DNA-damaging agents 1 . Although in bacterial systems proteins such as photolyase can directly reverse thymine dimers in DNA to restore two adjacent thymine bases, the only known human proteins that directly reverse DNA damage are the AlkB enzymes 2–4 and AGT (or MGMT). AGT directly repairs O 6 -alkylguanine lesions in DNA, which result from endogenous sources such as S-adenosylmethionine and environmental toxins. These lesions are mutagenic, causing GC-to-AT transition mutations, and cytotoxic, forming the basis of anticancer chemothera- pies that involve DNA methylation or chloroethylation to cause apopto- sis 5,6 . Hence, increased AGT levels confer alkylation resistance in human tumors 7 and inhibitors of AGT, such as O 6 -benzylguanine, are currently in clinical trials as anticancer therapeutics 8 . AGT repairs O 6 -alkylguanines by irreversibly transferring the alkyl lesion to an active site cysteine (Fig. 1a), and this unique, stoichiometric mechanism makes it particularly amenable to inhibition. Despite many years of intense effort on the structural biology of damage reversal proteins such as AGT, no structures have been pub- lished for any direct damage reversal proteins in complex with DNA. To address the major questions regarding how AGT finds, accesses and repairs O 6 -alkylguanine lesions in DNA and to aid in inhibitor design, we determined two structures of AGT bound to different DNA sub- strates by X-ray crystallography (Fig. 1). The first complex, an inactive C145S mutant bound to an O 6 -methylguanine-containing oligonu- cleotide (Fig. 1c), is a pretransfer complex of the predominant bio- logical substrate. In the second complex, active AGT is covalently crosslinked to an oligonucleotide containing the substrate analog N 1 ,O 6 -ethanoxanthosine (Fig. 1b,d), a mechanistic, crosslinking inhibitor that is analogous to DNA treated with the chloroethylating therapeutic agents 9 . These structures and biochemical data reveal a novel protein-DNA architecture, provide a detailed structural basis for the reaction mechanism and suggest novel mechanisms by which AGT finds and accesses damaged guanines in the context of genomic DNA. RESULTS Overall structure of the complex Crystals of the O 6 -methylguanine and ethanoxanthosine complexes diffract, respectively, to 3.2 Å and 3.3 Å (Table 1), and despite crystal- lizing in different space groups, have essentially identical structures, with a main chain r.m.s. deviation of 0.77 Å. Despite this moderate resolution, 2F o – F c and composite omit electron density maps (Figs. 1c,d and 2) show the DNA and protein clearly and define the AGT-DNA binding architecture and DNA conformational change unambiguously. The C-terminal domain of the two-domain α/β-fold houses the cysteine nucleophile in a conserved active site PCHR sequence adjacent to the DNA-binding HTH motif. The N-terminal domain is composed of a three-stranded β-sheet separated from two 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 J.T. (jat@scripps.edu). Published online 27 June 2004; doi:10.1038/nsmb791 DNA binding and nucleotide flipping by the human DNA repair protein AGT Douglas S Daniels 1 , Tammy T Woo 1,5 , Kieu X Luu 2 , David M Noll 3 , Neil D Clarke 3 , Anthony E Pegg 2 & John A Tainer 1,4 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. © 2004 Nature Publishing Group http://www.nature.com/natstructmolbiol