Strand-specific recognition of DNA damages by XPD provides insights into nucleotide excision repair substrate versatility

Abstract

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5'-3' helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a "final" verification state, which may then trigger the recruitment of further NER proteins.

Keywords: Atomic Force Microscopy; DNA Damage; DNA Helicase; DNA Lesion Recognition; DNA Repair; Protein-DNA Interactions; Single Molecule Biophysics.

FIGURE 1.

DNA binding by XPD. A, AFM image of XPD bound to circular pUC19N plasmid DNA (for example, arrows) in the presence of ATP. B, protein coverage of different DNA substrates in the presence (black) and absence (gray) of ATP: circular, pUC19N plasmid; linear substrates: nsp, linearized full-length pUC19N (2,729 bp) and 1,813-bp fragment; bubble, 916-bp fragment containing a 8-nt DNA bubble at ∼31% of the fragment length; F/bubble, 916-bp fragment containing a fluorescein (F) within the context of a DNA bubble at ∼31% of the fragment length. Protein coverage is normalized to the number of base pairs of the different DNA substrates. Additional AFM images for the different DNA substrates and ATP conditions are shown in supplemental Fig. S2. C, AFM image of XPD bound to linear F/bubble DNA, in the presence of ATP. Arrows indicate examples for specific (at ∼31% DNA length) and nonspecific XPD-DNA complexes in yellow and white, respectively, as well as for an end bound complex in red. D, XPD binding position distributions on F/bubble DNA substrate in the presence (black, n = 231) and absence (gray, n = 398) of ATP demonstrate stable complex formation at the lesion supported by ATP-dependent transitions (+ ATP) versus a minor binding preference at the unpaired DNA region (− ATP). A slight preference of binding to the DNA fragment ends is also apparent (enhanced occupancies at 0% DNA length). Fractional occupancies are plotted for ∼33-bp-long sections of 916-bp DNA from DNA fragment ends (0%) to 50% DNA length (at the DNA center). The scale bars in A and C correspond to 200 nm.

FIGURE 2.

DNA translocation and lesion recognition by XPD. Localization specificities of XPD in the presence (black bars) or absence (gray bars) of ATP or in the presence of ATPγS (white bars) were obtained from Gaussian fits (supplemental Fig. S5) to the statistical AFM position distributions of XPD on different DNA substrates (schematically indicated below the plot). Numbers for DNA substrates are also given, consistent with those in Table 3, which lists specificities for all targets and ATP conditions. Striped bars show specificities of the taXPD K170A variant for the fluorescein/5′ bubble DNA and CPD/3′ bubble DNA substrate, as indicated. The red circle represents a fluorescein, and the blue rectangle represents a CPD lesion. Significance (classed as p < 0.05 (*) and p < 0.01 (**)) was calculated for differences in specificity compared with a DNA bubble without a lesion in the presence of ATP (first bar).

FIGURE 3.

XPD-DNA complex conformation. The distributions of DNA bend angles induced by XPD at a fluorescein lesion in the context of a DNA bubble indicate conformational changes in the protein-DNA complexes in the presence of ATP (B) or ATPγS (C) compared with in the absence of ATP (A). A, in the absence of ATP, nonspecific bend angles (gray bars) and bend angles at the lesion site (specific; black bars) are similar and fit by a Gaussian curve centered at ∼50° (n_nsp = 174; n_spec = 113). B and C, nonspecific bend angles are not affected by the presence of either ATP or ATPγS (gray bars), whereas the specific bend angle distributions show a significant shift (p < 10⁻¹¹; see “Experimental Procedures” and Table 4) to an average bend angle of ∼65° in the presence of ATP or ATPγS (n_nsp,ATP = 94; n_spec,ATP = 137; n_nsp,ATPγS = 242; n_spec,ATPγS = 224). Specific bend angle distributions (DNA bending at the lesion site) are independent of the type of lesion (D, E, G, and H) or the presence or absence of a DNA bubble (further conditions in Table 4). D, fluorescein/5′ bubble; E, fluorescein/3′ bubble. G, CPD/5′ bubble. H, CPD/3′ bubble. A helicase hyperactive XPD variant (K170A) is unable to recognize the lesions and undergo conformational changes: wild-type XPD (A–E, G, and H) and K170A XPD (F and I) with fluorescein/5′ bubble (F) and CPD/3′ bubble (I) in the presence of ATP.

FIGURE 4.

XPD damage verification model. A, XPD-ssDNA complex model based on the crystal structure with partial ssDNA bound. The RecA-like helicase domains 1 and 2 are shown in yellow and red, respectively; the iron-sulfur cluster domain in cyan, and the arch domain is in green. The modeled ssDNA strand is shown in blue, and the backbone of the DNA originally resolved from the crystal structure is in orange. The positions of Lys¹⁷⁰ (blue, N), which is mutated in the helicase XPD variant (K170A), as well as the iron-sulfur cluster (red, iron; yellow, sulfur) are indicated. B, model of XPD damage verification for different lesions. XPD is loaded at a DNA bubble and translocates in 5′ to 3′ direction on the DNA (arrow). Panel I, translocation is stalled by a bulky lesion such as fluorescein (red circle) on the translocated strand, which acts as a mechanical road block to protein movement. Panel II, for an intrastrand pyrimidine dimer (CPD, blue rectangle), protein translocation is not majorly hindered by the presence of the lesion on the translocated strand, whereas an alternative lesion sensing mechanism, which has yet to be more thoroughly characterized, allows recognition of the lesion by XPD on the nontranslocated strand. It could be envisioned that XPD simultaneously exploits both types of lesion recognition and that the nature of the lesion determines which strategy becomes dominantly important and initiates repair competent conformational changes.