DNA damage recognition and repair by 3-methyladenine DNA glycosylase I (TAG)

Abstract

DNA glycosylases help maintain the genome by excising chemically modified bases from DNA. Escherichia coli 3-methyladenine DNA glycosylase I (TAG) specifically catalyzes the removal of the cytotoxic lesion 3-methyladenine (3mA). The molecular basis for the enzymatic recognition and removal of 3mA from DNA is currently a matter of speculation, in part owing to the lack of a structure of a 3mA-specific glycosylase bound to damaged DNA. Here, high-resolution crystal structures of Salmonella typhi TAG in the unliganded form and in a ternary product complex with abasic DNA and 3mA nucleobase are presented. Despite its structural similarity to the helix-hairpin-helix superfamily of DNA glycosylases, TAG has evolved a modified strategy for engaging damaged DNA. In contrast to other glycosylase-DNA structures, the abasic ribose is not flipped into the TAG active site. This is the first structural demonstration that conformational relaxation must occur in the DNA upon base hydrolysis. Together with mutational studies of TAG enzymatic activity, these data provide a model for the specific recognition and hydrolysis of 3mA from DNA.

Figure 1

The structure of the TAG–DNA complex. (A) The crystallographic model of TAG (blue ribbons; yellow HhH motif) bound to DNA (orange sticks) and 3mA (ball-and-stick). The abasic site in the DNA is highlighted in green and the coordinated Zn²⁺ ion is shown as a magenta sphere. (B) Schematic representation showing the electrostatic (dashed lines) and van der Waals (wavy lines) interactions between protein side-chain and main-chain (mc) atoms with the DNA.

Figure 2

Two conformations of the abasic DNA backbone. (A) Refined models showing the flipped (yellow) and stacked (pink) conformations of the THF abasic site are superimposed on an unbiased composite omit electron density map (contoured at 2σ). Only density corresponding to nucleic acid is shown for clarity. The proximity of the DNA to the 3mA (green sticks) is highlighted by a double-sided arrow. (B) The flipped and stacked DNA conformations are shown separately against a van der Waals surface representation of the protein. THF and 3mA carbon atoms are highlighted in green.

Figure 3

TAG interrogation of the DNA base stack. TAG binds the DNA damage site by intercalating side chains (blue) into both the lesioned (yellow) and non-lesioned (gold) DNA strands. Hydrogen bonds to the estranged thymine are shown as dashed lines and the 3mA base is colored green.

Figure 4

The nucleobase binding pocket of TAG. Protein residues are shown in gray, 3mA in green, DNA in yellow, and waters as red spheres. Hydrogen bonds are depicted as blue dashed lines.

Figure 5

Comparison of 3-methyladenine DNA glycosylases. Top: structure-based sequence alignment of TAG, AlkA, and MagIII shows the relative positions of residues important for DNA binding and base excision. TAG secondary structure elements are shown schematically, with the HhH motif colored yellow. Residues contacting the DNA backbone are boxed, intercalating plug (pink) and wedge (yellow) residues are highlighted, and side chains contacting the estranged base are labeled blue. Side chains confirmed (green) or postulated (gray) to contact 3mA in the base binding pocket are highlighted. Residues verified biochemically to affect substrate binding or catalysis are shown in boldface and the catalytic aspartates in AlkA and MagIII are shaded blue. TAG residues that coordinate Zn²⁺ are shaded orange. Bottom: crystal structures of TAG/DNA/3mA, AlkA/DNA, and MagIII/3mA (with modeled DNA) are shown. Protein solvent-accessible surfaces are colored according to the electrostatic potential (blue, positive; red, negative). An alternate version of this figure showing all HhH glycosylase/DNA complexes is available as Supplementary data.

Figure 6

Model of a TAG/3mA-DNA substrate complex. TAG side chains important for DNA intercalation (Gly43, Leu44) and 3mA binding (Glu38, Trp46, Tyr16, Gln41) are shown as green ball-and-sticks. See text for details.