Cell, Volume 135 Supplemental Data Structural Basis of UV DNA-Damage Recognition by the DDB1-DDB2 Complex

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1 Cell, Volume 135 Supplemental Data Structural Basis of UV DNA-Damage Recognition by the DDB1-DDB2 Complex Andrea Scrima, Renata Koníčková, Bryan K. Czyzewski, Yusuke Kawasaki, Philip D. Jeffrey, Regina Groisman, Yoshihiro Nakatani, Shigenori Iwai, Nikola P. Pavletich, Nicolas H. Thomä

2 Supplemental Tables Table S1: Data collection and refinement statistics for hsddb1-drddb2 complexes Data collection hsddb1-drddb2 14mer 6-4 photoproduct (DDB dr -DNA 6-4PP ) hsddb1-drddb2 16mer THF (DDB dr -DNA THF ) hsddb1-drddb2 DNA free X-ray source SLS X10SA PXII SLS X10SA PXII SLS X10SA PXII Space group P P P Cell parameters a=113,51 Å; b= Å ;c= Å a=113,21 Å; b= Å; c= Å a= Å; b= Å; c=173.49å Resolution (Å) ( ) ( ) ( ) Wavelength (Å) Completeness (%) 99.9 (99.9) 99.9 (99.9) 99.8 (99.7) Unique reflections (5526) (7344) (11488) Redundancy 7.4 (7.2) 7.3 (6.7) 6.2 (6.2) R sym (%) 7.0 (44.0) 5.1 (35.7) 7.0 (40.6) I/σI 23.4 (4.4) 27.4 (6.0) 19.0 (4.7) Refinement PDB code 3EI1 3EI2 3EI3 R work a (%) R free b (%) Reflections (work/free) (48302/2724) (62419/3506) (87271/4861) Rmsd Bond length (Å) Bond angle (deg) Ramachandran (%) -most favoured -additional allowed -generously allowed -disallowed No. of atoms (protein / DNA/ water / ligand) (11551/569/194/13) (11569/603/284/13) (11542/-/700/13) Avg. B-factor (Å 2 ) Values in parentheses are for last resolution shell a R work = h F o F c / h F o, where F o and F c are the observed and calculated structure factor amplitudes of reflection h. b R free is the same as R work, but calculated on the reflections set aside from refinement

3 Table S2: Data collection, phasing and refinement statistics for hsddb1-hsddb2 complexes Data collection hsddb1-hsddb2 DNA free hsddb1-hsddb2 31mer THF (DDB hs -DNA THF ) Data collection hsddb1-hsddb2 DNA free: KAu(CN) 2 hsddb1-hsddb2 DNA free: Thimerosal X-ray source APS 8BM APS 17IDB X-ray source APS 8BM APS 8BM Space group H32 C2 Space group H32 H32 Cell parameters a=b=268.5 Å c=471.4 Å a=252.1 Å b=85.9 Å c=260.3 Å β=113.6 Cell parameters a=b=269.1 Å c=478.1 Å a=b=269.7 Å c=481.1 Å Resolution (Å) ( ) ( ) Resolution (Å) ( ) ( ) Wavelength (Å) Wavelength (Å) Completeness (%) 91.0 (68.3) 86.4 (71.8) Completeness (%) 88.2 (82.3) 99.9 (99.8) Unique reflections (6575) (3148) Unique reflections (4562) (5231) Redundancy ) 2.7 (2.4) Redundancy 7.7 (6.0) 8.0 (6.7) R sym (%) 13.2 (48.3) 10.0 (47.8) R sym (%) 19.5 (42.6) 18.7 (45.2) I/σI 8.5 (1.7) 9.1 (3.7) I/σI 1.4 (3.8) 15.2 (5.2) Refinement Phasing PDB code 3EI4 - Resolution (Å) Resolution (Å) R work a (%) Phasing Power c R free b (%) R Cullis d Reflections (work/free) (76663/1994) (30884/1743) R Cullis d (anomalous) Rmsd Bond length (Å) Bond angle (deg) Mean FOM e Ramachandran (%) -most favoured -additional allowed -generously allowed -disallowed No. of atoms (protein / DNA/ water / ligand) (35256/-/-/-) (23504/2530/-/-) Avg. B-factor (Å 2 ) Values in parentheses are for last resolution shell a R work = h F o F c / h F o, where F o and F c are the observed and calculated structure factor amplitudes of reflection h. b R free is the same as R work, but calculated on the reflections set aside from refinement c Phasing power = [F 2 H(calc) / (F PH(obs) F PH(calc) ) 2 ] 1/2, where F PH(obs) and F PH(calc) are the observed and calculated derivative structure factors, respectively. d R cullis is the mean residual lack-of-closure error divided by the dispersive or anomalous difference. e Figure of Merit (FOM) = < P(α) exp(iα) / P(α)>, where P(α) is the probability distribution for the phase.

4 Table S3: Minor/major groove width for DDB dr -DNA 6-4PP Table S5: Phosphate-Phosphate distances for DDB dr -DNA 6-4PP Major groove Minor groove Base (D U+m; m=4) Width (Å) Base (D U+m; m=-3) Width (Å) C(D-5) T(U+1) G(D-4) A(U-1) C(D-3) A(U-2) G(D-2) C(U-3) A(D-1) G(U-4) 64(D+1) C(U-5) 64(D+2) G(U-6) G(D+3) G(U-7) C(D+4) G(U-8) G(D-2) G(U+5) A(D-1) C(U+4) 64(D+1) G(U+3) 64(D+2) C(U+2) G(D+3) T(U+1) C(D+4) A(U-1) G(D+5) A(U-2) C(D+6) C(U-3) C(D+7) G(U-4) C(D+8) C(U-5) Table S4: Minor/major groove width for DDB dr -DNA THF Damaged strand PP-distance (Å) PP-distance (Å) Undamaged strand D-5 D U+5 U+4 D-4 D U+4 U+3 D-3 D U+3 U+2 D-2 D U+2 U+1 D-1 D U+1 U-1 D+1 D U-1 U-2 D+2 D U-2 U-3 D+3 D U-3 U-4 D+4 D U-4 U-5 D+5 D U-5 U-6 D+6 D U-6 U-7 D+7 D U-7 U-8 Major groove Minor groove Base (D U+m; m=4) Width (Å) Base (D U+m; m=-3) Width (Å) A(D-7) T(U+3) A(D-6) T(U+2) T(D-5) A(U+1) G(D-4) T(U-1) A(D-3) T(U-2) A(D-2) T(U-3) T(D-1) C(U-4) -(D+1) G(U-5) A(D+2) T(U-6) A(D+3) C(U-7) G(D+4) C(U-8) G(D-4) T(U+7) A(D-3) T(U+6) A(D-2) A(U+5) T(D-1) C(U+4) -(D+1) T(U+3) A(D+2) T(U+2) A(D+3) A(U+1) G(D+4) T(U-1) C(D+5) T(U-2) A(D+6) T(U-3) G(D+7) C(U-4) G(D+8) G(U-5) Table S6: Phosphate-Phosphate distances for DDB dr - DNA THF Damaged PP-distance PP-distance Undamaged strand strand (Å) (Å) D-7 D U+7 U+6 D-6 D U+6 U+5 D-5 D U+5 U+4 D-4 D U+4 U+3 D-3 D U+3 U+2 D-2 D U+2 U+1 D-1 D U+1 U-1 D+1 D U-1 U-2 D+2 D U-2 U-3 D+3 D U-3 U-4 D+4 D U-4 U-5 D+5 D U-5 U-6 D+6 D U-6 U-7 D+7 D U-7 U-8

5 Figure S1: Sequence alignment and secondary structure assignment of DDB2 The alignment was generated by T-coffee (Notredame et al., 2000). Secondary structure assignment was based on the structures presented in this work. Swiss-Prot entry codes for sequences used: danio rerio (Q2YDS1); xenopus tropicalis (Q66JG1); chicken (Q5ZJL7); mouse (Q99J79); rat (NCBI: XP_242065); human (Q92466).

6 Conservation in zebrafish versus human DDB2: In the course of this project, a large number of deletion constructs of zebrafish and human DDB2, were tested for solubility, and where applicable, they were pursued in crystallization trials. In addition, 25 different oligonucleotide duplexes containing abasic site (THF) and 6-4 photoproduct (6-4PP) lesions were tested for co-crystallization. High resolution diffraction was only observed for the N- terminally truncated zebrafish DDB2 construct (residues 94 to 457) bound to 6-4PP or THF lesions, as described in the manuscript. Analogous boundaries for human DDB2 (residues 59 to 427) were extensively tested, but did not result in diffracting crystals. The overall conservation of zebrafish DDB2 versus its human ortholog are 55% (similarity)/37% (identity) for the full length constructs and 74% (similarity)/51% (identity) for the constructs used in crystallization trials. Out of the 17 residues in direct contact with DNA (see Figure 3E), 13 are identical. Out of the 23 residues in the DDB2 interface (with a cutoff around 3.9 Å) 8 are conserved (Supplemental Figure S1). The overall high conservation between zebrafish and human DDB2 is also evident when comparing the 3.3 Å structure of hsddb2 with that of the 2.3 Å drddb2 structure (rmsd 1.1 Å over 337 Cα-atoms). Overall, the underlying theme of damage recognition is highly conserved between zebrafish and human DDB2. Mutations in XP-E patients: On the basis of the DDB1-DDB2 structure, XP-E mutations can be subdivided into two mutually nonexclusive subcategories: (i) mutations that directly interfere with DNA binding, and (ii) mutations that impair DDB1 binding. In vitro, the solubility of DDB2 is highly dependent on the presence of DDB1 (A. Scrima & N. Thoma, unpublished), and this suggests that DDB2 mutations that affect DDB1 binding would indirectly interfere with lesion recognition by reducing the amount of functional DDB2 available in the cell. It is also possible that even when isolated DDB2 binds to the lesion, the lack of DDB1 and its associated ubiquitin ligase activity would result in a defective NER response. DDB1-DDB2 interface mutations: In XPE patient GM01389 a Leu350Pro mutation combined with a deletion of Asn349 (Leu387 and Asp386 in drddb2) impairs DDB1 binding and damage detection (Nichols et al., 2000; Rapic-Otrin et al., 2003). These residues are located on top of the DDB2-WD40 propeller involved at the interface with DDB1 (Asn349) and the packing between the DDB2-WD40 propeller and helix h2 (Leu350). The DNA binding surface is unaffected by these mutations. DNA binding mutations: The Asp-X-Arg motif, conserved among DCAF members, is present in DDB2 and a point mutation Arg273His (equivalent to Arg309 in drddb2) is found in patients XP2RO and XP3RO (Tang and Chu, 2002; Wittschieben and Wood, 2003). In the structures of DDB dr and DDB hs, this motif is solvent exposed, 6.3 Å away from the nearest DDB1 residue (R111), and is thus unlikely to play a direct role in DDB1 binding (Supplemental Figure S2). In accordance with this, in vitro studies show that DDB2 Arg273His remains bound to DDB1, while damage recognition is abolished (Chu and Chang, 1988; Wittschieben et al., 2005). The deleterious effect of the Arg273His mutation is likely via interfering with the structural integrity of blade 4, part of which forms the DNA binding interface.

7 The Lys244Glu charge reversal seen in XP-E patient XP82TO strongly diminishes overall DNA damage binding with the DDB1-DDB2 interaction not being disturbed (Rapic-Otrin et al., 2003; Wittschieben et al., 2005). Lys244 (Lys280 in drddb2) is located directly in the DNA binding interface. This residue is in contact with the phosphate backbone close to the lesion site (D +3 ; D +4 ). A mutation of residue Asp307Tyr (Asp344 in drddb2) is seen in patient XP25PV. This mutation severely disrupts damage detection and also diminishes the extent of complex formation with DDB1 (Rapic-Otrin et al., 2003; Rapic Otrin et al., 1998). Asp307 is located on blade 5 and stabilizes the transition from strand B to C. The mutation is expected to interfere with the folding of blade 5 and possibly of the entire WD40 domain, indirectly affecting binding to DNA and to DDB1. Figure S2: The conserved DxR-motif in DDB2 (A) The conserved DxR and DxH motifs are solvent exposed and not involved in DDB1-DDB2 interactions. (B) Close-up view of the DxR-motif highlighting the ion pair of Arg309 with Asp307 and the stacking interaction with the conserved Trp292. Mutation of Arg309 (Arg273 in hsddb2) could potentially destabilize these interactions and thereby compromise the integrity of blade 4. The BPA and BPC domains of DDB1 are shown in red and yellow, respectively. DDB1-CTD domain is shown in lightgray. DNA is shown in black and gray (damaged and undamaged strand). DDB2 is shown in green with the conserved DxR and DxH motifs shown as stick model in gray. (C) Alignment of DDB2 with the motifs highlighted in red.

8 Figure S3: DDB1-DDB2 interaction and implications for DCAF architecture (A) Close-up view of the helix-loop-helix motif of DDB2 mediating binding to DDB1. Residues of the conserved interaction motif 104 SI(L/V)(H/R) LG 115 are shown in stick representation. Surface of DDB1 is shown in gray, the remaining colors are as in Supplemental Figure S2. (B) Overlay of the helix-loop-helix motif of DDB2 (green) and the simian virus 5 V protein (blue) bound to DDB1. (C) Sequence alignment of hepatitis B virus protein X and DDB2 highlighting the sequence similarity in helix h1. (D) Sequence alignment of WDR23 and DDB2 covering helix h1 and helix h2. (E) GST-pulldown using human HBV-X (HBV-X L :A76-E125; HBV-X S :A76-S101) as bait and His-DDB1 as prey. Proteins were expressed in insect cells as N-terminal fusion proteins, with HBV-X as single-expression (lanes 1 and 4), and coexpressed with DDB1 (lanes 2 and 3). Both constructs bind to DDB1, with HBV-X L binding more effectively to DDB1. The band labelled as GST corresponds to endogenous insect cell Glutathion S-transferase. (F) His-pulldown using His-tagged human WDR23 (WDR23 L : G88-Q546; WDR23 S : R155-Q546) as bait with GST-DDB1 as prey. WDR23 L, containing helix h1 and helix h2, forms a stoichiometric complex with DDB1 (lane 2). Deletion of the helical segment in WDR23 S (lane 3) abolishes DDB1 binding resulting in background levels of unspecifically bound DDB1 (control, lane 1). For lane 3 the 4fold amount was loaded to adjust for levels of bound WDR23.

9 The DDB1-DCAF architecture: DDB1 binds to a family of 30 DDB1-CUL4 associated factors (DCAFs). These proteins function as DDB1-CUL4 substrate adaptors in a variety of different cellular processes including repair, replication, transcription and signaling (reviewed in Lee and Zhou, 2007; O'Connell and Harper, 2007; Petroski and Deshaies, 2005). Epitopes within DDB2 helix h1 and the subsequent loop (drddb2: 104 SI(L/V)(H/R) LG 115 ) contain two highly conserved motifs involved in DDB1 binding (Supplemental Figure S3A). A similar DDB1 binding motif has also been identified by Hanaoka and colleagues (Fukumoto et al., 2008). The position of helix h1 is structurally reminiscent to that of SV5 bound to DDB1 (Li et al., 2006) (Supplemental Figure S3B). Despite little sequence identity both helices superimpose with an overall rmsd of 2.01Å over 10 residues. Studies have identified a peptide in the unrelated hepatitis B virus X protein (HBV-X) required for DDB1 binding. This epitope also shows sequence similarity to DDB2 helix h1 and is sufficient to mediate binding to DDB1 (Supplemental Figures S3C, E) (Bergametti et al., 2002). Inspection of the DCAF family using a hidden Markov model (HMM) identified a helical segment immediately preceding the WD40 domain in WDR23/DCAF11 (Supplemental Figure S3D). Deletion of these two predicted helices results in diminished DDB1 binding (Supplemental Figure S3F). A similar effect is seen upon deletion of helix h1/helix h2 in DDB2 (A. Scrima & N. Thoma unpublished and Jin et al., 2006). Besides WDR23, we were not able to reliably identify further DDB2-like DCAF proteins. While this might be partially due to low primary sequence conservation of helix h1, there likely is a second architectural group of DDB1 binding proteins for which the WD40 domain alone is sufficient for DDB1 binding (Groisman et al., 2003; Han et al., 2006; Lee and Zhou, 2007; Schuetz et al., 2006).

10 Figure S4: Electron densities of the 6-4 photoproduct and the abasic site (A-B) Close-up view of the 6-4 pyrimidine-pyrimidone photodimer (A) and the abasic site mimic THF (B) with final 2F O - F C electron density contoured at 1σ shown in blue. (C-D) Composite omit map (CNS, Brunger et al., 1998) shown in green, with the 6-4 pyrimidine-pyrimidone dimer (C) and the abasic site mimic THF (D), contoured at 0.8σ and 0.6σ, respectively. The DNA is shown as stick representation with the damaged strand shown in black/orange and the undamaged strand in gray/purple (carbon/phosphate atoms).

11 Figure S5: DNA-kinking angle of the 31mer THF complex structure Overlay of the 14mer 6-4PP, 16mer THF and 31mer THF complex structures. In all three structures the observed kinking angle corresponds to approx. ~40.

12 Figure S6: DNA-damage binding in the context of chromatin (A-B) Structure of DDB hs DNA THF (A) overlaid with that of the nucleosome (B) (PDB: 1AOI). The 23 bp DNA fits best with the lesion dimer at position 51/52 (rmsd: 2.44 Å). Shifting the lesion dimer position by +/- 1 bp results in a slightly increased rmsd of 4.61 and 3.33 Å, respectively. (C) Model of the DDB hs DNA THF -nucleosome in complex with CUL4A/RBX1-E2 (corresponding to position 2 in Figure 7). Color scheme: DDB1-BPA, red; DDB1-BPB: magenta; DDB1-BPC, yellow; DDB1-CTD: lightgray; DDB2, green; DNA THF damaged/undamaged strand are in black and gray, respectively. CUL4A/RBX1: gray; E2: darkgray with the active site shown as spheres in red. Histones are colored as indicated in the figure insert. The nucleosomal-dna is shown in orange. Only the 23bp used for the overlay are shown for DNA THF. The phosphodiester backbone of DDB2-bound DNA can be fitted on that of nucleosome-bound DNA with an rmsd of ~ Å over 23 bp (including the THF-lesion and its two flanking 11 bp segments) with the CPD approximately at positions 0/1, 10/11, 20/21, 31/32, 41/42, 51/52 or 62/63 (Davey and Richmond, 2002).

13 Figure S7: Model of simultaneous damaged DNA binding by XPC and DDB2 Model of Rad4/XPC bound to the DDB hs DNA THF complex. XPC binds to the undamaged dsdna segment through the TGD-BHD1 domain thereby positioning the BHD2-BHD3 domains to the damaged site. Color scheme: DDB2, green; TGD, orange; BHD1, magenta; BHD2, cyan; BHD3, red; DNA THF damaged/undamaged strand are in black and gray, respectively. Rad23 has been omitted for clarity. Model for DDB2-mediated recruitment of XPC to the site of damage In this model, the XPC/Rad4 BHD2-BHD3 domains would clash minimally with the DDB2-bound dsdna 5 to the lesion, but this clash would be largely alleviated if the XPC/Rad4 BHD2-BHD3 domains adopt the conformation seen in the apo-rad4 structure. The possibility of a DDB2-DNA- XPC complex is supported by the demonstration that XPC/Rad4 has substantial affinity for undamaged dsdna that resides in its TGD-BHD1 domain (Maillard et al., 2007; Min and Pavletich, 2007), and by the recent report that XPC-RAD23B interacts with the DDB1-DDB2 complex (Sugasawa et al., 2005). Such an interaction may facilitate the recruitment of XPC to the DDB2-bound DNA.

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