Volume 59, Issue 6, Pages 1025-1034 (September 2015) Tripartite DNA Lesion Recognition and Verification by XPC, TFIIH, and XPA in Nucleotide Excision Repair  Chia-Lung Li, Filip M. Golebiowski, Yuki Onishi, Nadine L. Samara, Kaoru Sugasawa, Wei Yang  Molecular Cell  Volume 59, Issue 6, Pages 1025-1034 (September 2015) DOI: 10.1016/j.molcel.2015.08.012 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 59, 1025-1034DOI: (10.1016/j.molcel.2015.08.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Purification of TFIIH and Core7 (A) An illustration of TFIIH complex (Zhovmer et al., 2010) with Core7 colored yellow and CAK blue. (B) Purified TFIIH and Core7 (5 μg each) were analyzed on a 4%–12% Bis-Tris NuPAGE SDS gel and stained with Coomassie blue. (C) Samples of Core7 purification steps (5 μg each), crude extract (1), and eluent from Heparin (2), M2 beads (3), HisTrap (4), Superose 6 (5), are shown with the molecular weight markers (M) on a 4%–12% SDS-PAGE gel. Molecular Cell 2015 59, 1025-1034DOI: (10.1016/j.molcel.2015.08.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 ATPase Activity of TFIIH and Core7 (A) ATPase activities of TFIIH and Core7 were assayed alone or in the presence of 50-fold molar excess of ssDNA (20 nt) or dsDNA (25 bp) (Table S1) and shown in the bar graph. (B) ATPase activities of Core7 in the presence of ssDNA, dsDNA, or bubble-structured DNA of variant lengths. (C) ATPase activities of WT, XPBKR, XPDKR, and double KR (2KR) mutant Core7 as protein alone or with different ssDNAs and different bulky lesions. (D) ssDNA, with or without an AP lesion, stimulated the Core7 ATPase equally. Standard deviations were estimated based on at least triplicates. Molecular Cell 2015 59, 1025-1034DOI: (10.1016/j.molcel.2015.08.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 EMSA of DNA Binding by Core7 (A) Core7 has different affinity for ssDNA (45 nt, ss45) and dsDNA (45 bp, ds45). The 5′-32P-labeled DNAs (0.5 nM) were incubated with Core7 (0, 0.25, 0.5, 1, 2, 4, 8, 16, and 20 nM). Two protein-DNA complexes were detected with ss45 (1 and 2). (B) Different lengths of 32P-labeled ssDNAs (0.5 nM) were incubated with Core7 (0, 0.5, 4, and 20 nM). The longer the ssDNA used, the more the protein-DNA complexes formed. (C) The same EMSA assays as shown in (A) were performed for ssDNA of variant lengths, and the results were quantified and plotted. Dissociation constants (KD) were calculated by fitting the EMSA data with the non-linear regression model (Prism) and shown in the table next to the plot. Error bars were estimated from triplicate measurements. (D and E) Effects of Cy5 (D), CisPt (E), and ATP on DNA binding by Core7. Core7 (0.5, 1, 4, and 20 nM) and ssDNA were incubated with or without ATP and MgCl2 and analyzed by EMSA. The percentage of Core7-DNA complexes in each lane was quantified and indicated below the gels. Molecular Cell 2015 59, 1025-1034DOI: (10.1016/j.molcel.2015.08.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Bulky Lesions on the 5′ Overhang Strand Impede the Core7 Helicase Activity (A) DNA unwinding activities of Core7 on a normal 5′ overhang DNA (N_45/35), with Cy5 on the top strand (Cy5_TOP), a mismatched base pair (M_45/35), or Cy5 on the bottom strand (Cy5_BOT). DNA substrates are diagramed at the bottom of each panel, and asterisks indicate 5′-32P labels. Core7 concentrations were 10, 20, 40, and 80 nM in each assay. Triangles (▵) indicate the heat-denatured substrates, and the “C” stands for control of DNA substrate alone. (B) Effects of CisPt on the Core7 helicase activity were compared by placing the lesion CisPt on the top (Pt_TOP) or the bottom strand (Pt_BOT) of the normal DNA duplex N_44/34 or N_54/44, respectively, each with a 5′ overhang (Table S2). (C) DNA unwinding activities of Core7 with an AP lesion on the top (AP_TOP) or bottom strand (AP_BOT). (D) Unwinding of DNA with a 3′ overhang by Core7 was undetectable. The Cy5 near the ds-ss junction promoted DNA unwinding, perhaps by melting DNA locally and generating a free 5′ end. (E) The XPDKR mutant Core7 had very weak helicase activity and was inhibited by CisPt present on the strand scanned by XPD. To detect the weak activity, protein concentrations were increased to 80–320 nM. (F) The XPBKR mutant Core7 had slightly reduced helicase activity compared with WT. (G) Relative activities of WT Core7 in unwinding DNAs with a 5′ overhang. Helicase activities (%) were normalized to the corresponding undamaged DNA substrates and plotted in the same colors as DNA diagrams shown in (A)–(C). Standard deviations were estimated based on triplicates. Molecular Cell 2015 59, 1025-1034DOI: (10.1016/j.molcel.2015.08.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Inhibition of the Core7 Helicase Activity by Bulky Lesion and XPA (A) XPA significantly enhanced Core7 helicase activities on a normal DNA (N_44/34), but had little stimulatory effect when CisPt was present on the top strand scanned by XPD (Pt_TOP). (B) XPA had no stimulatory effect when CisPt was present on the bottom strand (Pt_BOT) either. (C) AP lesions also inhibited the stimulatory effect of XPA on the Core7 helicase. Consistent results were obtained in multiple experiments with 1 or 2 nM DNA, and the latter is shown. Relative helicase activities of Core7 in the presence of increasing amounts of XPA are plotted beneath each gel in (A)–(C). (D and E) Comparison of DNA unwinding by Core7 alone or Core7 and XPA together at 1:1 molar ratio with or without DNA lesion. CisPt was on the strand scanned by XPD (D) or the complementary strand (E), and XPA enhances lesion-dependent stalling of Core7 by ∼2-fold in both cases. Relative helicase activities are shown in the bar graph beneath. Error bars were estimated from triplicate measurements. Molecular Cell 2015 59, 1025-1034DOI: (10.1016/j.molcel.2015.08.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Effects of CAK on DNA Binding, Unwinding, and Dual Incision (A) TFIIH, WT, and two mutant Core7 were assayed for their ability to activate dual incisions in the reconstituted NER reaction with a 6-4 photoproduct (6-4 PP). The amount of each protein was equalized based on anti-XPB immunoblotting (IB). (B) EMSA of ssDNA binding by TFIIH and WT Core7 (0, 0.25, 0.5, 1, 2, 4, 8, and 16 nM). The black arrow indicates protein-DNA complexes of Core7 and TFIIH. (C) DNA helicase activities of Core7 and TFIIH in the presence and absence of XPA. (D) EMSA examination of Core7 and TFIIH recruitment by XPC/RAD23B/Centrin-2 to a preformed DNA bubble (bubble7, Table S1). DNA complexes with XPC, TFIIH, or Core7 are marked. Supershift indicates the XPC-TFIIH-DNA and XPC-Core7-DNA complexes. Molecular Cell 2015 59, 1025-1034DOI: (10.1016/j.molcel.2015.08.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 A Model of Tripartite Lesion Recognition and Verification (A) TFIIH is associated with chromatin weakly in a non-specific manner without DNA lesion. (B) In the presence of DNA lesions (red hexagon), TFIIH is loaded onto the DNA by XPC or RNA Pol II in a strand- and orientation-specific manner. It scans both DNA strands in parallel with XPD and XPB helicases. (C) Translocation of TFIIH on normal DNA is accelerated by XPA. Small base lesions and mismatches (small red triangle), which are substrates for base excision and mismatch repair, respectively, behave like normal DNA. (D) Upon detection of a bulky lesion (red star) on the strand scanned by XPD, both XPB and XPD helicases are stalled by the lesion. XPA enhances the stalling and hence bulky-lesion recognition. After dissociation of CAK, Core7, RPA, and XPA stabilize the bubble structure around the lesion and recruit XPF and XPG to make sequential dual incisions. Repair-coupled DNA synthesis can facilitate incision by XPG and lesion removal. Molecular Cell 2015 59, 1025-1034DOI: (10.1016/j.molcel.2015.08.012) Copyright © 2015 Elsevier Inc. Terms and Conditions