Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair - PubMed (original) (raw)

Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair

Chenxu Zhu et al. Proc Natl Acad Sci U S A. 2016.

Abstract

NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)-a preferred substrate-for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzyme-promoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction.

Keywords: QM/MM; base-excision repair; enzyme catalysis; glycosylase; substrate recognition.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Crystal structures of NEIL1 bound to dsDNA containing THF and Tg, respectively. (A) Overall view of NEIL1 bound to a THF-containing duplex, with the flipped THF shown in purple. (B) Accommodation of THF in the active site of NEIL1. Density map (2_F_obs − _F_cal) of THF is shown. Hydrogen bonds are shown in green dashed lines. The red dashed line shows the distance between the α-amino group of Pro2 and C1 atom of THF. (C) Overall view of NEIL1 bound to a Tg-containing duplex, with the flipped Tg shown in purple. (D) Overlay of the apo and THF-bound NEIL1 structures, highlighting the conformational change of Arg242. In the Tg-bound structure, Arg242 in the flexible lesion recognition loop flips over, and the polar side chain of Arg242 points to the Tg base. Tyr244 resides approximately in the same location in both apo (gray) and Tg-bound (green) structures. (E) Overlay of the lesion recognition in the apo (gray), Tg (green)-, and THF (cyan)-bound structures. The same angle as in D is shown here for comparison.

Fig. 2.

Fig. 2.

Tautomerization-dependent recognition of Tg. (A) Electron density map of Tg. The blue 2_F_obs − _F_cal map is contoured at 1.2σ and the green _F_obs − _F_cal omit map—by removing the 5-methyl (C7) and 6-hydroxyl group (O21) of Tg—is contoured at 3.0σ [omit maps removing 5-hydroxyl group (O22) are shown in SI Appendix, Fig. S3_A_]. (B) The active-site pocket of Tg-bound NEIL1. Hydrogen bonds are shown in green dashed lines. The hydrophobic pocket surrounding the 5-methyl group of Tg is indicated by a yellow curve. The N3 of Tg and Nη of Arg242 is highlighted with yellow background. (C) Tg tautomers in the lactam (Upper) and lactim (Lower) forms. (D) Optimized structure of the Tg-bound NEIL1 active site. Due to the Tg2-OH tautomer, a new hydrogen bond was observed between 2-OH of Tg and Glu6. Key distances are marked in black (in angstroms), and the 2-OH group of Tg is highlighted with yellow background.

Fig. 3.

Fig. 3.

Proposed mechanisms and computational verification. (A) Ribose-protonated pathway initiated with the NEIL1–R242–Tg2-OH structure. Along this pathway, we list the reactant (R2-1), two intermediate states (R2-2 and R2-3), and the product (R2-4). (B) The quantitative characterization of the above proposed mechanisms using QM/MM umbrella sampling (see SI Appendix, Detailed Materials and Methods, Section IX for more details): NEIL1–R242–Tg2-OH ribose-protonated pathway (full line), and NEIL1–R242–Tg4-OH ribose-protonated pathway (SI Appendix, Scheme S1) (dashed line). Numbers along the curves correspond to relative free energies of the transition states and intermediate states (including reactant and product).

Fig. 4.

Fig. 4.

Structure of unedited NEIL1 (Lys242) bound to Tg. (A) Overall view of unedited NEIL1 bound to dsDNA containing Tg. Lys242 is highlighted in a red circle. (B) The active-site pocket of unedited NEIL1 bound to Tg. Hydrogen bonds are shown in green dashed lines. The N3 of Tg and Nζ of Lys242 is highlighted with yellow background. (C) Superposition of unedited (green) and edited (gray) NEIL1–Tg structures. The positions of the flipped base are almost identical, and both Arg242 and Lys242 point toward Tg. (D) Optimized structure of the Tg-bound NEIL1 (Lys242) active site. A hydrogen bond between 2-OH of Tg and Glu6 was also observed in this structure. Key distances are marked in black (in angstroms), and the 2-OH group is highlighted with yellow background.

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