Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex

Abstract

DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 "opens" up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion "opening" is slow (˜5-10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise "twist-open" mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins.

Keywords: DNA damage recognition; DNA unwinding dynamics; temperature-jump perturbation; time-resolved fluorescence spectroscopy; xeroderma pigmentosum.

Fig. 1.

DNA conformational rearrangements during lesion recognition by Rad4 probed by tC^o/tC_nitro. (A) Structures of an ideal B-form DNA (Left) and Rad4-bound–specific lesion recognition (open) complex (Right; Protein Data Bank ID code 2QSH) (54). The gray rotation arrow indicates the direction of DNA unwinding upon opening. The DNA-binding domains of Rad4 are shown: TGD-BHD1-BHD2 (lime green), BHD3 (red), and the β-hairpin within BHD3 (blue). In the open complex, the flipped-out nucleotides on the undamaged strand (black) directly contact Rad4, whereas the flipped-out nucleotides on the damaged strand (magenta) do not and become disordered (dotted magenta line). (B) Chemical structures of tC^o and tC_nitro and Watson–Crick type base pairing of tC^o with a guanine (G). (C) FRET efficiency between the tC^o and tC_nitro incorporated within normal B-DNA is plotted as a function of the distance between the two probes. FRET decreases as the distance increases, but additionally depends on the relative orientations of the absorption and emission dipoles of the fluorophores. B and C are adapted with permission from ref. ; copyright (2009) American Chemical Society.

Fig. 2.

AN12 and AN12u DNA constructs. (A) Models of DNA duplexes when in B-DNA conformation (Left) and in the open complex (Right), in the same orientations as in Fig. 1A. The positions are marked for the mismatched nucleotides (black and red) and the FRET probes, tC^o (donor, cyan) and tC_nitro (acceptor, blue) of AN12. (B) DNA sequences of AN12 and AN12u. D, donor; P, acceptor. (C) FRET at 25 °C in free AN12 (pink, n = 11), AN12–Rad4 (magenta, n = 4), free AN12u (lime, n = 18), and AN12u–Rad4 (green, n = 9).

Fig. 3.

Equilibrium and T-jump measurements on AN12 bound to Rad4. (A and B) I_DA as a function of temperature for AN12–Rad4 (magenta, n = 4) and AN12 (pink, n = 11); the corresponding I_D is in black. I_D and I_DA have been normalized to match at the lowest temperature. The intensities at 25 °C measured before and after the heating/cooling cycle are both indicated. T-jump relaxation traces are shown for AN12_DA in the presence/absence of Rad4 (C and D) and for AN12_D in the presence/absence of Rad4 (E and F). Only AN12_DA–Rad4 in C exhibits kinetics in the T-jump time-window (7.7 ± 0.5 ms after a T-jump from 19–26 °C), whereas D–F exhibit only the slow T-jump recovery after a similar T-jump. (G) Relaxation rates of AN12–Rad4 (pink) vs. the inverse of the final temperature (after the T-jump). The different symbols indicate three independent sets of measurements. The continuous pink line is an Arrhenius fit to the relaxation rates, yielding an activation enthalpy of 18.5 ± 1.1 kcal/mol. The dashed black line is an Arrhenius fit to the relaxation rates on 2AP-labeled mismatch DNA (AN3) bound to Rad4, from a study by Chen et al. (49). A schematic representation of the mismatched AN12_DA construct design is also shown. a.u., arbitrary unit.

Fig. 4.

Equilibrium and T-jump measurements on AN12 bound to Rad4 β-hairpin mutants. (A and B) Equilibria I_DA for AN12–Δβ-hairpin3 (orange, n = 4) and for AN12–ΔBHD3 (red, n = 4) are shown vs. temperature, with the corresponding I_D in black. (C) T-jump relaxation traces with AN12_DA–Δβ-hairpin3 show a single phase: 89 ± 7.4 μs after a T-jump from 18–25 °C. (D) AN12_DA–ΔBHD3 shows two phases: 131 ± 12 μs and 5.8 ± 0.3 ms after a T-jump from 26–32 °C. (E) Relaxation rates of AN12–Δβ-hairpin3 (orange triangles) and AN12–ΔBHD3 (red diamonds for fast phase, red squares for slow phase) vs. inverse temperature from two independent sets of measurements (open/filled symbols). The continuous lines are Arrhenius fits to the rates, with activation enthalpies of 11.9 ± 0.3 kcal/mol for AN12–Δβ-hairpin3 and 3.3 ± 2.4 kcal/mol (fast phase) and 27.5 ± 32.4 kcal/mol (slow phase) for AN12–ΔBHD3. The pink and dashed black lines are from Fig. 3G.

Fig. 5.

AN14 and AN14u DNA constructs. DNA models (A) and sequences (B) are shown for AN14 and AN14u, as in Fig. 2 A and B. (C) FRET at 25 °C in free AN14 (light purple, n = 12), AN14–Rad4 (purple, n = 6), free AN14u (cyan, n = 13), and AN14u–Rad4 (blue, n = 7).

Fig. 6.

Equilibrium and T-jump measurements on AN14 and AN14u bound to WT Rad4. (A and B) Equilibria I_DA for AN14–Rad4 (purple, n = 5) and for AN14u–Rad4 (blue, n = 4) are shown vs. temperature with the corresponding I_D in black. T-jump relaxation traces with AN14_DA–Rad4 (C) and AN14u_DA–Rad4 (D) show relaxation times of 603 ± 35 μs and 575 ± 114 μs, respectively, after a T-jump from 15–22 °C. (E) Relaxation rates of AN14–Rad4 (purple) and AN14u–Rad4 (blue) vs. inverse temperature from two independent sets of measurements (open/filled symbols). The continuous lines are Arrhenius fits to the rates, with activation enthalpies of 18.2 ± 2.2 kcal/mol (AN14–Rad4) and 11.6 ± 3.3 kcal/mol (AN14u–Rad4). The pink, orange, red, and dashed black lines are from Fig. 4E. A schematic representation of the nonspecific mismatched AN14_DA construct design is also shown.

Fig. 7.

Conformational trajectory of lesion recognition by Rad4. (A) Three distinct binding modes for Rad4/XPC as it searches for, interrogates, and recognizes a damaged site and the time scales for fluctuations between these modes are shown. (B, Top) A free-energy profile that may underlie the observed kinetics along the recognition trajectory. Damaged DNA in nonspecific binding modes is shown with inherently higher free energy than undamaged DNA due to the damaged/induced destabilization. The 100- to 500-μs twisting step is depicted with a smaller energetic barrier than the 5- to 10-ms rate-limiting distortion step (‡), which is followed by rapid β-hairpin insertion and full-nucleotide flipping. (B, Bottom) Putative diffusional landscapes of the protein along DNA are illustrated along coordinates orthogonal to the conformational trajectory. As the recognition proceeds, the diffusional landscape gets progressively rough; once the β-hairpin is inserted, the protein is practically obstructed from diffusing away, and thus recognition.