DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy

Abstract

O(6)-Alkylguanine-DNA alkyltransferase (AGT) is a crucial target both for the prevention of cancer and for chemotherapy, since it repairs mutagenic lesions in DNA, and it limits the effectiveness of alkylating chemotherapies. AGT catalyzes the unique, single-step, direct damage reversal repair of O(6)-alkylguanines by selectively transferring the O(6)-alkyl adduct to an internal cysteine residue. Recent crystal structures of human AGT alone and in complex with substrate DNA reveal a two-domain alpha/beta fold and a bound zinc ion. AGT uses its helix-turn-helix motif to bind substrate DNA via the minor groove. The alkylated guanine is then flipped out from the base stack into the AGT active site for repair by covalent transfer of the alkyl adduct to Cys145. An asparagine hinge (Asn137) couples the helix-turn-helix DNA binding and active site motifs. An arginine finger (Arg128) stabilizes the extrahelical DNA conformation. With this newly improved structural understanding of AGT and its interactions with biologically relevant substrates, we can now begin to unravel the role it plays in preserving genetic integrity and discover how it promotes resistance to anticancer therapies.

Fig. 1

AGT substrates. (A) Normal guanine:cytosine base pair (left), O⁶-methylguanine:cytosine base pair (middle), and O⁶-methylguanine:thymine base pair (right). (B) O⁶-methylguanine, O⁶-benzylguanine, and O⁴-methylthymine.

Fig 2

The AGT active site and proposed reaction mechanism. (A) Hydrogen bond network and proposed reaction mechanism for AGT. To facilitate attack at the O⁶-alkyl carbon, His146 acts as a water-mediated general base to deprotonate Cys145. The resultant imidazolium ion is stabilized by Glu172. Tyr114 donates a proton to N3 of O⁶-methylguanine. (B) Stereo view of the X-ray crystallographic structure of the AGT C145S-DNA complex (pdb 1t38) showing the active site, O⁶-methylguanine substrate, and hydrogen bond network.

Fig. 3

Sequence alignment of AGTs from select organisms. The active site motif and the DNA-binding region of the helix-turn-helix motif are outlined in blue. Human AGT numbering is used. The arginine finger (128), “Asn hinge” (137), and active site cysteine (145) are highlighted in yellow.

Fig. 4

Selected AGT pseudosubstrates.

Fig. 4

Selected AGT pseudosubstrates.

Fig. 5

Human AGT X-ray crystallographic structure, zinc site, and DNA binding. (A) Unreacted AGT structure (pdb 1eh6). The N-terminal domain is shown in green, the C-terminal domain is shown in yellow, and the HTH motif is shown in blue. The active site cysteine, arginine finger, “Asn hinge”, and zinc ligands are shown in ball-and-stick representation. (B) DNA-bound AGT structure (pdb 1t39). AGT uses the recognition helix of the HTH motif to bind the minor groove of DNA.

Fig. 6

Human AGT substrate binding and nucleotide flipping. X-ray crystallographic structures of AGT in complex with DNA substrates containing (A) O⁶-methylguanine (pdb 1t38), (B) N¹,O⁶-ethanoxanthosine (pdb 1t39), and (C) N⁴-p-xylylenediaminecytosine (pdb 1yfh). Tyr114 and Arg128 may promote 3′ phosphate rotation-induced nucleotide flipping.