Volume 22, Issue 1, Pages 27-37 (April 2006) Conserved XPB Core Structure and Motifs for DNA Unwinding: Implications for Pathway Selection of Transcription or Excision Repair  Li Fan, Andrew S. Arvai, Priscilla K. Cooper, Shigenori Iwai, Fumio Hanaoka, John A. Tainer  Molecular Cell  Volume 22, Issue 1, Pages 27-37 (April 2006) DOI: 10.1016/j.molcel.2006.02.017 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 XPB Conserved Sequence, Secondary Structure, Motifs, and Structural Architecture (A) Sequence conservation between human XPB and AfXPB proteins. Top, schematic alignment between AfXPB (Af) and human XPB (Hs). Bottom, sequence alignment between the full-length AfXPB (residues 1–452, Af) and the corresponding core region (residues 243–686) of human XPB (Hs). Every tenth residue is marked by dots. Based on the AfXPB structure, shown are the β strands S1–S20 (S→), helices H1–H11 (αααα), undefined residues in the full-length AfXPB structure (dots), identical residues (bold letters), conserved helicase motifs I-VI (highlighted colors), the N-terminal DRD similar to MutS MRD (blue frame), HD2 insertion ThM with conserved positive charges (purple frame), and the RED motif (red diamonds and letters). N-AfXPB sequence is indicated by cyan arrows and C-AfXPB by green arrows under the sequence. (B–D) N-AfXPB, full-length AfXPB, and C-AfXPB structures revealing motifs, secondary structures, and domains displayed in ribbons. (B) Stereo pair showing the MutS-like DRD (blue) joins helicase domain HD1 (cyan). (C) Full-length AfXPB showing the hinge joining HD1 and HD2, the RED motif side chains, plus the architectural arrangement of the domains and motifs with the DRD and conserved motifs colored as in (A) with short helix 310 elements not indicated in (A) also displayed. (D) Stereo pair of C-AfXPB showing HD2 (green) and protruding helical polymerase-like thumb domain (purple, ThM) that is partly disordered in full-length AfXPB. Molecular Cell 2006 22, 27-37DOI: (10.1016/j.molcel.2006.02.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 DNA Binding and Helicase Activities of Truncated and Full-Length AfXPB Constructs (A) SDS-PAGE results for purified AfXPB (F), N-AfXPB (N), and C-AfXPB (C) expressed in bacteria. Protein markers (M) are also shown. (B and C) Electrophoresis mobility shift assay. Two micrograms bovine serum albumin (BSA) and AfXPB proteins (F, N, C) were each tested for binding to ssDNA and dsDNA substrates at 50°C. Five micrograms of each protein was used for assays on dsDNA containing tetrahydrofuran (THF) or 5-hydroxycytosine (5-OH-C) at 37°C. Free ssDNA (ss) and dsDNA (ds) as well as protein-DNA complexes (com) are labeled, respectively. (D) Helicase activities with different substrates. Lanes 1, 6, and 8, ssDNA control; lanes 2 and 3, dsDNA with a 5′ overhang; lanes 4 and 5, dsDNA with a 3′ overhang; lane 7, blunt-ended dsDNA; lanes 9 and 10, blunt-ended dsDNA with one strand containing CPD (bar); lanes 11 and 12, blunt-ended dsDNA with one strand containing (6-4) photoproduct (PP) (bar); and lanes 2, 4, 9, and 11, no AfXPB control. The asterisk (∗) indicates P-32 labeling. (E) Helicase assays of AfXPB mutants on dsDNA with a 3′ overhang. Lanes marked D and S contain ds and ssDNA controls, respectively. The full-length AfXPB (wt) protein was used at two different amounts: 1 μg (lane marked 20) or 50 ng (lane marked 1). The singly substituted mutant proteins R210A and E211A and the triply-substituted mutant protein R210A / E211A / D212A (AAA) were used at 1 μg. Sequence alignments (bottom) at helicase Motif III and RED motif are shown for XPB homologs from archael (Af), yeast (Sc), fly (Dm), and human (Hs). The yeast motifs are underlined. Molecular Cell 2006 22, 27-37DOI: (10.1016/j.molcel.2006.02.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Proposed Interaction of AfXPB N-Terminal DRD with Kinked dsDNA (A) The AfXPB-DRD structure (residues 24–83, blue) is superimposed with the MutS mismatch recognition domain (orange residues A31–A98) in complex with DNA (orange, PDB code 1EWQ) with marked N- and C-terminal ends and sequence similar elements encoded (bottom). (B) Stereo pair of the N-AfXPB structure (gray) with the DRD (blue), RED motif (red), Motif I-III&Ia (cyan), and the kinked DNA (orange) position based upon the superposition with the MutS-DNA complex. Molecular Cell 2006 22, 27-37DOI: (10.1016/j.molcel.2006.02.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Proposed Conformational Change in AfXPB upon Binding DNA, Based on Comparison of the Full-Length AfXPB Structure with the HCV NS3 Helicase in Its ssDNA Bound Conformation For the observed open full-length AfXPB structure (left; color-coded ribbons as in Figures 1B and 1D), when HD1 is superimposed with HCV NS3 helicase domain 1 (right; HCV in gold; PDB code 1A1V), the orientation of AfXPB HD2 differs from its counterpart in the HCV NS3 helicase, corresponding to a ∼170° rotation at the glycine-containing hinge joining HD1 and HD2 to form the proposed active, closed conformation of XPB (right). Molecular Cell 2006 22, 27-37DOI: (10.1016/j.molcel.2006.02.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Structural Comparison of XPB HD2 Insertion ThM with the Thumb Domain of T7 DNA Polymerase and Taq DNA Polymerase (A) Stereo pair of C-AfXPB insertion ThM (residues 252–313, purple) superimposed over the T7 DNA polymerase thumb domain (PDB code 1T7P, residues A243–A251, 340–351, and 374–396, gold) and Taq DNA polymerase thumb domain (PDB code 1TAQ, residues 460–526, gray). (B) Positively charged residues at the groove between HD2 (gray) and ThM (purple). Helicase motifs IV–VI are highlighted in green. Conserved positively charged residues between AfXPB and human XPB are underlined. Molecular Cell 2006 22, 27-37DOI: (10.1016/j.molcel.2006.02.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Proposed Structure-Based Mechanism whereby Damage Verification by XPB Promotes Unwinding of Damaged dsDNA for NER (A) Schematic model shows how XPB DRD (blue), HD1 (cyan), RED motif (red), HD2 (green), and ThM (purple) may be activated by dsDNA (yellow) containing a DNA damage site (orange). AfXPB initially binds to damaged DNA in an open conformation. DNA damage verification by N-XPB (DRD and HD1) induces the rotation of C-XPB (HD2/ThM) to form the closed helicase-DNA complex, facilitated by HD1-mediated ATP hydrolysis that aids dsDNA melting at the lesion (orange) to allow the RED loop residues (red) to intrude between the opened DNA strands. The red arrow indicates the direction in which the RED motif unzips the base pairs. (B) The predicted closed AfXPB conformation consisting of structural domains DRD (blue), HD1 (cyan), and HD2 (green) together with motifs RED (red sphere side chains) and ThM (purple). (C) A proposed AfXPB-DNA complex. The AfXPB surface is mapped with the color-coded electrostatic potential (red, negative; white, neutral; and blue, positive). The DNA (purple phosphate backbone with bases as gold bars) was taken from the helicase Pcr-DNA complex (PDB code 3PJR) connected to a thymine-dimer-containing DNA from the photolyase-DNA complex (PDB code 1TEZ). Molecular Cell 2006 22, 27-37DOI: (10.1016/j.molcel.2006.02.017) Copyright © 2006 Elsevier Inc. Terms and Conditions