Volume 37, Issue 2, Pages 235-246 (January 2010) UV-Induced Association of the CSB Remodeling Protein with Chromatin Requires ATP- Dependent Relief of N-Terminal Autorepression  Robert J. Lake, Anastasia Geyko, Girish Hemashettar, Yu Zhao, Hua-Ying Fan  Molecular Cell  Volume 37, Issue 2, Pages 235-246 (January 2010) DOI: 10.1016/j.molcel.2009.10.027 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 UV Irradiation Induces Stable CSB-Chromatin Association (A) Flow chart of the protein fractionation protocol used. (B) Temporal analysis of CSB partitioning in MRC5 cells after UV irradiation. Western blots were probed with anti-N-terminal CSB and anti-GAPDH antibodies. Total core histones were visualized by Ponceau S staining. (C) Quantification of CSB levels in soluble versus chromatin-containing fraction. Experiments were performed in triplicate, and data are shown as means with standard errors. (D) ChIP-western analysis of CSB-chromatin association. Anti-histone H3 antibodies were used to purify chromatin from crosslinked MRC5 cells. Chromatin-associated proteins were resolved by SDS-PAGE and analyzed by western blot, using anti-CSB and anti-histone H3 antibodies. “-” represents protein A beads-only control. Bands of higher molecular weight likely represent CSB modifications. (E) UV dose-response analysis of CSB partitioning in MRC5 cells. Antibodies used for western blot analysis were as indicated. (F) CSB protein levels in (E) were quantified as in (C). Molecular Cell 2010 37, 235-246DOI: (10.1016/j.molcel.2009.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Some CS-Associated CSB Mutations Disrupt UV-Induced Chromatin Association and ATPase Activity (A) Schematic representation of the CSB protein with the positions of four mutations found in CS patients. Striped boxes represent the seven conserved helicase motifs, and gray boxes represent two potential nuclear localization sequences. A and G represent acidic and glycine-rich regions, respectively. (B) CS1AN-Sv cells stably expressing wild-type or mutant CSB proteins were irradiated with a series of UV doses. CSB partitioning was determined by western blot analysis. Lower-molecular-weight bands in the soluble fractions result from minor degradation due to overexpression. (C) Densitometric quantitation of the data in (B). Experiments were performed in triplicate, and data are shown as means with standard errors. (D) Coomassie-stained gel showing proteins used. (E) ATPase assays of CSB and CSBR670W ad CSBW851R. (F) Determination of the effects of V957G and P1042L on DNA-stimulated ATPase activity of CSB. ATPase rates were measured in the presence of different DNA concentrations over a 3-fold dilution series. The highest DNA concentration is 30 ng/μl (light blue). The descending concentrations of DNA are shown as light blue, orange, yellow, light green, green (+), dark green, black, red, and blue (no DNA control). CSB, CSBV957G, and CSBP1042L were used at 4.9, 4.8, and 3.5 nM, respectively (see also Figure S1). Molecular Cell 2010 37, 235-246DOI: (10.1016/j.molcel.2009.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Effects of CSB ATPase Activity on UV-Induced CSB-Chromatin Association (A) Schematic representation of CSB mutant proteins used in assays. (B) Coomassie-stained gel showing proteins used. (C) ATPase reactions of CSB and CSBR894E. (D) KM of wild-type and mutant CSB proteins for DNA was determined by plotting ATPase rates against DNA concentrations. Data are shown as means with standard errors (n ≥ 3). (E) kcat, the maximal rate constant of ATPase under conditions of saturating ATP/Mg2+ and DNA, as indicated. Data are shown as means with standard errors (n ≥ 3). (F) Partitioning of CSBwt and mutant CSB proteins transiently expressed in CS1AN-Sv cells. Cells were irradiated with UV light (100 J/m2) and proteins were fractionated 1 hr after UV irradiation, as in Figure 1. Molecular Cell 2010 37, 235-246DOI: (10.1016/j.molcel.2009.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Primary Structural Determinants Directing CSB-Chromatin Association (A) Schematic representation of mutants used in assays. (B and C) Experiments were performed by transiently expressing the indicated proteins in the CS1AN-Sv cell line, as described in Figure 3F. (B) Partitioning of CSB deletion proteins CSB-N, CSB-M, or CSB-C. (C) Partitioning of the deletion proteins CSBΔC and CSBΔN. (D) ChIP-western analysis of CSBΔN-chromatin association. Anti-histone H3 antibodies were used to purify chromatin from crosslinked CS1AN-Sv cells stably expressing CSBΔN, as in Figure 1D. (E) Coomassie-stained gel showing mutant CSB proteins used in Figures 4 and 5. (F and G) Graphs illustrate the DNA affinities, KM (F) and ATP hydrolysis activity, kcat (G) of CSB deletion mutants. Data are shown as means with standard errors (n ≥ 3). N.D., not determined. Asterisk represents no stimulation over background (see also Figure S2). Molecular Cell 2010 37, 235-246DOI: (10.1016/j.molcel.2009.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 CSB-N Inhibits the ATPase Activity of CSBΔN In Trans (A) CSBΔN (3 nM) was used in ATP hydrolysis assays in the absence (black) or presence (colored) of DNA (1.1 ng/μl), with varying amounts of CSB-N (blue: 0 nM, dark green: 0.2 nM, light green: 0.9 nM, orange: 3.8 nM, and red: 15 nM). (B) Same as in (A), except 3 nM BRG1 was used instead of CSBΔN. (C) Same as in (A), except 9 nM CSB was used instead of CSBΔN. (D and E) ATPase activities of CSBΔN and CSBΔN + CSB-N. CSBΔN and CSB-N were used at 3 and 7 nM, respectively, in the presence of varying amount of DNA (3-fold serial dilutions). The highest DNA concentration is 30 ng/μl (light blue). The descending concentrations of DNA are shown as light blue, orange, yellow, light green, green (+), dark green, black, red, and blue (no DNA control). (F) ATPase rates determined from (D and E) were plotted against DNA concentration to determine KM and Vmax. Data are shown as means with standard errors (see also Figure S3, Table S1). Molecular Cell 2010 37, 235-246DOI: (10.1016/j.molcel.2009.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 ATP Hydrolysis Is Not Required for CSBΔN-Chromatin Association (A and B) CSBR670WΔN, CSBW851RΔN, CSBV857GΔN, CSBK538AΔN, CSBK979EΔN, and CSBN653IΔN were transiently expressed in CS1AN-Sv and processed as in Figure 3F. The distribution of CSB was determined by western blot analysis. (C) ChIP-western analysis of CSBK538AΔN-chromatin association. Anti-histone H3 antibodies were used to purify chromatin from crosslinked CS1AN-Sv cells stably expressing CSBK538AΔN, as in Figure 1D. Molecular Cell 2010 37, 235-246DOI: (10.1016/j.molcel.2009.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 A Model for the Regulation of CSB-Chromatin Association Under conditions of normal cell growth or in the absence of lesion-stalled transcription, the interaction of CSB with chromatin is dynamic. Here, the ATPase domain (M) and the N-terminal region (N) primarily mediate the association of CSB with chromatin. The N-terminal region sequesters a DNA interaction surface (pink patch) within the C-terminal region (C) and promotes nonreactive chromatin association (bidirectional arrows). Transient CSB-chromatin interactions can occasionally lead to stimulation of ATPase activity, which induces a conformational change (open conformation) that repositions the N-terminal region and exposes residues in the C-terminal region (pink patch), normally occluded by the N-terminal region. (A) In the absence of lesion-stalled transcription, this open conformation is short lived and converts back to the closed conformation, a process that might also permit CSB translocation as suggested for other ATP-dependent chromatin remodelers (Cairns, 2007). (B) In the presence of lesion-stalled transcription (red X), the open conformation is trapped by interactions between the N-terminal region and components of lesion-stalled transcription. CSB-chromatin association would then be stabilized by extending the chromatin contact surface from the ATPase domain to include residues in the C-terminal region (see also Figures S4). Molecular Cell 2010 37, 235-246DOI: (10.1016/j.molcel.2009.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions