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 damage at the O(6)-position of guanine in a unique, stoichiometric reaction. Crystal structures of AGT homologs from the three kingdoms of life reveal that despite their extremely low primary sequence homology, the topology and overall structure of AGT has been remarkably conserved. The C-terminal domain of the two-domain, alpha/beta fold bears a helix-turn-helix (HTH) motif that has been implicated in DNA-binding by structural and mutagenic studies. In the second helix of the HTH, the recognition helix, lies a conserved RAV[A/G] motif, whose "arginine finger" promotes flipping of the target nucleotide from the base stack. Recognition of the extrahelical guanine is likely predominantly through interactions with the protein backbone, while hydrophobic sidechains line the alkyl-binding pocket, as defined by product complexes of human AGT. The irreversible dealkylation reaction is accomplished by an active-site cysteine that participates in a hydrogen bond network with 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 release of repaired DNA and a signal for the observed degradation of alkylated AGT.
Daniels
D S
DS
The Skaggs Institute for Chemical Biology, Department of Molecular Biology, MB-4, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037-1027, USA.
Tainer
J A
JA
D000478:Alkylation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry; D001120:Arginine / Q000737:chemistry / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D019143:Evolution, Molecular; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D019077:Helix-Turn-Helix Motifs; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D019843:Archaeal Proteins; D001426:Bacterial Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D001120:Arginine; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003545:Cysteine