Conserved structural motifs governing the stoichiometric repair of alkylated DNA by O-6-alkylguanine-DNA alkyltransferase

O-6-alkylguanine-DNA alkyltransferase (AGT) directly repairs alkylation dam age at the O-6-position of guanine in a unique, stoichiometric reaction. Cr ystal structures of AGT homologs from the three kingdoms of life reveal tha t despite their extremely low primary sequence homology, the topology and o verall structure of AGT has been remarkably conserved. The C-terminal domai n of the two-domain, alpha/beta fold bears a helix-turn-helix (HTH) motif t hat has been implicated in DNA-binding by structural and mutagenic studies. In the second helix of the HTH, the recognition helix, lies a conserved RA V[A/G] motif, whose "arginine finger" promotes flipping of the target nucle otide from the base stack. Recognition of the extrahelical guanine is likel y predominantly through interactions with the protein backbone, while hydro phobic sidechains line the alkyl-binding pocket, as defined by product comp lexes of human AGT. The irreversible dealkylation reaction is accomplished by an active-site cysteine that participates in a hydrogen bond network wit h invariant histidine and glutamic acid residues, reminiscent of the serine protease catalytic triad. Structural and biochemical results suggest that cysteine alkylation opens the domain-interfacing "Asn-hinge", which couples the active-site to the recognition helix, providing both a mechanism for r elease of repaired DNA and a signal for the observed degradation of alkylat ed AGT. (C) 2000 Elsevier Science B.V. All rights reserved.