Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition

Abstract

Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination.

Figure 1.

(A) Comparison of structure at the wedge insertion site for simulation structures of Bst-Fpg–DNA complex with Phe113 (pink) or Trp113 (colored by atom) wedge. (B) Experimental (jagged traces) and fit (smooth curves) time courses of Trp fluorescence during the interaction of Eco-Fpg-F110W and Eco-Fpg-WT with G-ligand (G:C) and oxoG-substrate (oxoG:C). A.u., arbitrary units. (C) Kinetic simulation of accumulation and disappearance of various enzyme–substrate complexes during the interaction of Eco-Fpg-F110W with undamaged (G-ligand) and damaged (oxoG-substrate) DNA.

Figure 2.

Comparison of free energy profiles for lesion eversion with Phe (A) and Ala (B) wedge. In each curve, the intrahelical position is on the left. Subtracting the profiles (C) shows that the mutation stabilizes the intrahelical state.

Figure 3.

(A) Structure of duplex DNA containing a spontaneously everted AP site. (B and C) Experimental (jagged traces) and fitted (smooth curves) time courses of Trp (B) or aPu (C) fluorescence changes during cleavage of the oxoG:C (B and C) or AP substrates (C) by Eco-Fpg-WT and Eco-Fpg-F110A. A.u., arbitrary units.

Figure 4.

FRET time courses during cleavage of oxoG-substrate by Fpg. (A) Comparison of Cy3/Cy5 (red) and fluorescein/DABCYL time courses for Eco-Fpg-WT. (B) FRET time courses for Eco-Fpg-WT and Eco-Fpg-F110A. Jagged traces represent experimental data, smooth curves show the fit to Scheme VII (Supplementary Material). A.u., arbitrary units.

Figure 5.

(A) Structure overlap at the interrogation site of Bst-Fpg-F113A (colored by atom) and Bst-Fpg-WT (pink). (B and C) Color-coded difference in interaction energies due to the wedge, mapped onto the WT intrahelical (B) and extrahelical average structures (C). The figure shows the difference in per residue energy decomposition between WT and F113A mutant. Positive (red) values reflect more favorable interactions in the mutant.