Structural basis of UV DNA-damage recognition by the DDB1-DDB2 complex

Abstract

Ultraviolet (UV) light-induced pyrimidine photodimers are repaired by the nucleotide excision repair pathway. Photolesions have biophysical parameters closely resembling undamaged DNA, impeding discovery through damage surveillance proteins. The DDB1-DDB2 complex serves in the initial detection of UV lesions in vivo. Here we present the structures of the DDB1-DDB2 complex alone and bound to DNA containing either a 6-4 pyrimidine-pyrimidone photodimer (6-4PP) lesion or an abasic site. The structure shows that the lesion is held exclusively by the WD40 domain of DDB2. A DDB2 hairpin inserts into the minor groove, extrudes the photodimer into a binding pocket, and kinks the duplex by approximately 40 degrees. The tightly localized probing of the photolesions, combined with proofreading in the photodimer pocket, enables DDB2 to detect lesions refractory to detection by other damage surveillance proteins. The structure provides insights into damage recognition in chromatin and suggests a mechanism by which the DDB1-associated CUL4 ubiquitin ligase targets proteins surrounding the site of damage.

Figure 1. Overall structure of the DDB1-DDB2-DNA complex

(A) Ribbon representation of the DDB_dr–DNA^6-4PP complex: DDB2, green; DDB1-BPA, red; DDB1-BPB, magenta; DDB1-BPC, yellow; DDB1-CTD, gray; DNA^6-4PP damaged/undamaged strand in black/gray. (B) Ribbon representation of the DDB_dr–DNA^6-4PP complex rotated by 90° about the vertical axis relative to (A). (C) Schematic representation of hsDDB1 and drDDB2 with domain boundaries.

Figure 2. DDB1-DDB2 induced DNA-kinking upon complex formation and location of DNA-binding residues

(A) Superposition of the DDB_dr–DNA^6-4PP and DDB_dr–DNA^THF complex structures, highlighting the induced kink of ∼40° at the site of damage. DDB2, gray; DNA^6-4PP, blue; DNA^THF, red. (B) Cartoon representation of the DDB2_dr-WD40 β-propeller with DNA⁶-^4PP. Hairpin residues contacting the DNA are shown as yellow spheres; residues contacting the DNA-backbone are shown in orange. Bound DNA is shown in black/gray (carbon atoms) with phosphates in orange/purple for the damaged/undamaged strand, respectively. 2F_o-F_c electron density (contoured at 1.0 σ) for the DNA is shown in blue. (C) Cartoon representation of DDB2_dr in rainbow colors depicting the WD40 domain nomenclature. Blades are labelled 1-7 with the individual β-strands of blade 4 labelled with A-D. The backbone of the damaged and undamaged DNA strands is shown in black and gray, respectively, with the damaged 6-4 photoproduct shown as stick model.

Figure 3. Mechanism of 6-4 photodimer recognition

(A) Close-up of the DDB2 hairpin insertion (green) at the lesion with the damaged and undamaged strand depicted in yellow and brown, respectively. (B) Interaction of DDB2 with the DNA^6-4PP-backbone. The backbone of both strands is contacted by an array of positively charged residues crucial for the stabilization of the phosphate backbone compression at the damaged site (D₊₁, D₊₂). Parts of the DNA are omitted for clarity. (C) Close-up of the photodimer binding pocket stabilizing the flipped-out dinucleotide. Contacting residues are shown as stick models in yellow. The pyrimidine ring D₊₁ and the pyrimidone ring D₊₂ are shown in black and gray, respectively. Parts of the DNA have been omitted for clarity. (D) Chemical structure of the 6-4 pyrimidine-pyrimidone dimer. (E) Schematic representation of interactions between drDDB2 and DNA^6-4PP (with colors as in (A) and (B)).

Figure 4. Mechanism of abasic site recognition

(A) Interaction of DDB2 with the DNA^THF-backbone showing essentially identical interactions despite of a different sequence and type of damage. Parts of the DNA are omitted for clarity. (B) Sequence of DNA^THF used for co-crystallization. Disordered bases are shown faded. (C) Chemical structure of the abasic site mimic (THF) and the neighboring Adenosine.

Figure 5. Predicted binding mode of dewar 6-4PP and CPD in the damage binding pocket

(A) The 6-4 photodimer in the damage binding pocket with the pyrimidine carbon atoms shown in black and the pyrimidone carbon atoms in gray. (B) Overlay of the 6-4 PP (shown faded) with the Dewar isomer shown in yellow. (C) Chemical structure of the Dewar isomer. (D) The abasic site mimic (THF) with the neighboring Adenine in the damage binding pocket. (E) Overlay of the abasic site damage (shown faded) with a CPD shown in cyan. The covalently linked bases of the CPD can easily be accommodated in the damage binding pocket without sterical clashes. (F) Chemical structure of the CPD.

Figure 6. The DDB2-surface and hairpin are important structural features of the damage recognition process

(A) Conserved basic residues mapped onto the DDB2 surface (gray). Residues framed with a red box are in direct contact with the DNA phosphate backbone. The remaining 11 residues, located more distally (> 5.5 Å), form a second layer of arginines, lysines and histidines potentially guiding and pre-orienting the DNA towards DDB2 through long-range charged interaction. The ³⁷¹FQH³⁷³-hairpin motif is shown in yellow. (B) Overlay of the DDB2 hairpin residues ³⁷¹FQH³⁷³ from DNA-free (yellow) the DNA^6-4PP (blue) and DNA^THF (red) bound complex structures indicating that the hairpin acts as a rigid unit.

Figure 7. Implications for the role of UV-DDB mediated damage recognition in the context of chromatin

In structures of DDB1, DDB1-CUL4A/RBX1 and that of the DDB1-DDB2 complexes, the B-domain, which serves as the main attachment site for CUL4, is found in 8 different orientations (1. PDB:2HYE; 2. PDB: 2B5L; 3. DDB_hs–DNA^free; 4. DDB_hs–DNA^THF (Mol. A); 5. DDB_hs–DNA^THF (Mol. C); 6. DDB_dr-DNA^free; 7. DDB_dr–DNA^THF/6-4PP; 8. PDB:2B5M). The position of the E2 enzyme (UbcH5A; PDB: 2C4P) in relation to CUL4A/RBX1 was modeled using the c-Cbl-UbcH7 structure (PDB: 1FBV) as template. (A) Model of the DDB1-DDB2-DNA-CUL4A/RBX1-E2 complex (CUL4A/RBX1: lightgray; E2; darkgray). Numbers along the dotted path depict positions of the active site of the E2 (red spheres) based on the BPB-domain orientation in 8 different DDB1-structures. (B) As in (A) but rotated by 90° horizontally. (C-D) Different inclination angles of the BPB-domain tilt the CUL4 ligase by ∼20°, and confer a rotational movement of ∼ 120°. The CUL4/E2 samples ∼120° in a zone of ∼35-60 Å from the damage.