Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme

Abstract

How living systems detect the presence of genotoxic damage embedded in a million-fold excess of undamaged DNA is an unresolved question in biology. Here we have captured and structurally elucidated a base-excision DNA repair enzyme, MutM, at the stage of initial encounter with a damaged nucleobase, 8-oxoguanine (oxoG), nested within a DNA duplex. Three structures of intrahelical oxoG-encounter complexes are compared with sequence-matched structures containing a normal G base in place of an oxoG lesion. Although the protein-DNA interfaces in the matched complexes differ by only two atoms-those that distinguish oxoG from G-their pronounced structural differences indicate that MutM can detect a lesion in DNA even at the earliest stages of encounter. All-atom computer simulations show the pathway by which encounter of the enzyme with the lesion causes extrusion from the DNA duplex, and they elucidate the critical free energy difference between oxoG and G along the extrusion pathway.

Figure 1. Generation and recognition of 8-oxoguanine

a, Structural comparison of G versus oxoG, with differences highlighted. b, Overall structure of a lesion-recognition complex, LRC3, with an extrahelical oxoG lesion bound in the enzyme active site. c, Sequence-matched interrogation complex, IC3 (ref. 14), with a fully intrahelical target G•C base-pair. d, Close-up view of the oxoG-capping loop (OCL) and its contacts to oxoG in LRC3. The light pink region represents the residues deleted in EC3–5. Residues with grey side chains were mutated to prolines in the ECs bearing OCL point mutations. e, Sequence-matched encounter complex, EC3, bearing a fully intrahelical target oxoG•C base-pair.

Figure 2. Helix-penetration by MutM residues

a, LRC3; b, IC3; c, EC3. Colour-coding is as in Fig. 1, except side chains of the key residues (M77, R112, F114 and E78) are shown in cyan.

Figure 3. OxoG-dependent local DNA structure alterations at the site of the target base in EC3 and EC5

a and d, target G; c and f, target oxoG; b and e, models constructed by the addition of a carbonyl oxygen to the 8-position of the corresponding target G residue in a and d, respectively. The perspectives were chosen to facilitate examination of sugar puckers (diagrammed below a, c, d, and f). Steric clashes in the models are indicated by double-headed arrows.

Figure 4. Free energy profiles of nucleobase extrusion imply active participation of MutM

a, Free energies along the base extrusion pathways, as a function of the normalized arc length, Z, in b and Supplementary Fig. 17. Critical events associated with main energetic barriers are marked. b, Free energy landscape plots for extrusion of oxoG starting from EC4 (top) and G from IC4 (bottom). See Supplementary Fig. 16 for definitions of q_ext and q_rotat. The black dotted lines denote the minimal free energy paths for base extrusion, which define Z. The lowest free energy points in the intrahelical states are set to zero.

Figure 5. R112-catalysed oxoG extrusion

a –f, Snapshots from targeted molecular dynamics (TMD) simulations in the vicinity of the target base, showing helix invasion by R112 and concomitant extrusion of the target oxoG. The three DNA helix-invading MutM residues (M77, R112 and F114) are coloured in green. Nucleosides around the target oxoG•C pair are shown in gold. The inset figure of each panel is an orthogonal view. See the text for the progression of events a–f. Refer to the Supplementary Movies for the complete base extrusion trajectories of oxoG and G by MutM.