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ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor - PubMed

ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor

E Citterio et al. Mol Cell Biol. 2000 Oct.

Abstract

The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.

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Figures

FIG. 1
FIG. 1

CSB introduces negative supercoils in plasmid DNA upon binding. (A) Purified recombinant CSB and CSBK538R proteins. Aliquots of the Mono Q fractions of both proteins were separated by SDS-PAGE (8% acrylamide) and visualized by silver staining. In addition to the major CSB band, two degradation products (as determined by Western blotting) were visible. ∗, keratins which were also present in empty lanes. Molecular size markers are indicated. (B) Shift in the topoisomers distribution upon CSB binding. L, linear DNA (lane 1). Singly nicked (N) plasmid DNA (100 ng) (lane 2) was incubated in the presence of ATP alone (lanes 3 and 7) or with increasing amounts (20, 40, and 80 ng) of CSB (lanes 4, 5, and 6, respectively) or CSBK538R (lanes 8, 9, and 10, respectively). The DNA molecules were closed by the addition of E. coli DNA ligase, and the topoisomers were resolved by electrophoresis on a 1% agarose gel containing 0.5 μg of chloroquine per ml.

FIG. 2
FIG. 2

Mononucleosome remodeling by CSB. (A) Mononucleosome remodeling by CSB is ATP dependent. End-labeled nucleosome particles (approximately 3 ng of total nucleosomes) were incubated in the presence of ATP alone (lane 1) or with increasing amounts (10, 20, and 40 ng) of CSB in the presence (lanes 2, 3, and 4, respectively) or absence (lanes 5, 6, and 7, respectively) of ATP. Similar reactions were performed with CSBK538R in the presence of ATP (lanes 8 to 10). Remodeling was assessed by DNase I digestion. Filled arrows, sites of enhanced cutting due to the presence of CSB; open arrows, sites of reduced cleavage. N, naked control DNA (lane 11). (B) Nucleosome remodeling by CSB is stable upon removal of ATP by apyrase. Reactions were performed as described for panel A, and reaction mixtures contained 40 ng of CSB alone (lane 1) or with ATP (lane 2); where indicated, [γ-S]ATP (ATPγS) (2 mM) or apyrase (1 U) was added. [γ-S]ATP did not support remodeling (lane 3). Similarly, addition of apyrase (1 U) prior to CSB inhibited nucleosome remodeling (lane 4). However, addition of apyrase (1 U) after CSB had been present for 45 min did not inhibit or reverse nucleosome disruption, as shown by DNase I digestion after 2 min (lane 5), 15 min (lane 6), and 40 min (lane 7). As a control, nucleosomes were incubated in the presence of ATP alone (lane 8). N, naked DNA (lane 9). Arrows and filled arrows are as defined for panel A. (C) CSB nucleosome remodeling pattern is very similar but not identical to the one generated by the hSWI/SNF complex. Reactions were performed as described for panel A and reaction mixtures contained ATP alone (lane 1), 60 ng of CSB alone (lane 2) or with ATP (lane 3), or 300 ng of the isolated hSWI/SNF complex with ATP (lane 4) or alone (lane 5). Sites of enhanced (filled arrows) and of decreased (open arrows) DNase I digestion are indicated. Double-headed arrows between lanes 3 and 4 represent cleavage sites that distinguish the two nucleosome disruption activities. Bar, approximate position of the nucleosomal dyad axis. N, naked control DNA.

FIG. 3
FIG. 3

Remodeling of plasmid chromatin by CSB. (A) Remodeling of nucleosome arrays detected by MNase digestion. CSB induces loss of the regular nucleosome repeat characteristic of Drosophila-reconstituted chromatin in an ATP-dependent manner. A schematic outline of the assay is shown at left. Sarkosyl-stripped chromatin (40 ng) was incubated either in the absence of proteins (lanes 2 and 3) or with CSB (lanes 4 and 5), CSBK538R (lanes 6 and 7), or hSWI/SNF (lanes 8 and 9) in the presence of ATP (as described in Materials and Methods). Nucleosome organization was studied by MNase digestion (8 U), performed for 60 and 120 s, respectively. The positions of mononucleosomes (m), dinucleosomes (d), and trinucleosomes (t) are indicated by arrows and by dots. The DNA size marker (M) represents a ladder of 123-bp repeats (lane 1). (B) Remodeling of nucleosome arrays visualized as ATP-dependent changes in supercoiling. CSB, unlike hSWI/SNF, does not induce visible changes in the topology of nucleosomal plasmid DNA. Nucleosomal template was incubated with topoisomerase I alone (lanes 1 and 6) or with either hSWI/SNF (200 ng in lane 2; 20, 60 and 200 ng, respectively, in lanes 3, 4, and 5) or increasing amounts of CSB (threefold increments starting from 2.9 ng in lanes 7 and 12), in the presence (lanes 7 to 11) or absence (lanes 12 to 16) of ATP. The molar ratio of CSB (in lane 11) to the ATPase subunits of hSWI/SNF (in lane 5) was approximately 20:1, as determined by silver staining (not shown). Sc, supercoiled; R, relaxed or partially supercoiled.

FIG. 4
FIG. 4

Gel-shift analysis of CSB mononucleosome remodeling reactions. CSB does not catalyze the complete dissociation of histone octamers from DNA upon nucleosome binding and remodeling. Mononucleosomes (lanes 1 to 10) or free DNA (lanes 11 to 16) were incubated with increasing amounts of CSB (10, 20, 30, 60, and 100 ng for lanes 2, 3, 4, 5, and 6, respectively, and for lanes 12, 13, 14, 15, and 16, respectively; 60 and 100 ng, respectively, in lanes 7 and 8 and 9 and 10) and reactions were performed as described for those whose results are shown in Fig. 2, with (+) or without (−) ATP. Reaction mixtures were analyzed directly on native polyacrylamide gels or, where indicated, were treated with excess of cold competitor plasmid DNA before being loaded (lanes 7–10) (see Materials and Methods). Multiple DNA-protein complexes are visible (verticle bar).

FIG. 5
FIG. 5

CSB directly interacts with purified HeLa core histones. IP analysis was performed on reaction mixtures containing purified CSB and core histones (lanes 1 and 2, respectively, in panels A and B) (see Materials and Methods). After extensive washing, the antibody-bound protein complexes were separated on SDS–16.5% and –8% polyacrylamide gels to visualize the histones and CSB, respectively. Gels were stained with silver. (A) IP was carried out with anti-CSB antibodies. Analysis of the beads by SDS-PAGE showed that all four core histones specifically coimmunoprecipitate with CSB (lane 3) and not in a mock IP (lane 4). (B) IP with anti-histone, pan antibodies is shown. CSB is present in the bound fraction together with the histones (lane 3). No nonspecific binding is detected in the mock IP (lane 4).

FIG. 6
FIG. 6

CSB binds histone proteins in far-Western analysis. Samples of BSA (lanes 1), cytochrome c (lanes 2), and H1-depleted HeLa polynucleosomes (untreated [+ tails] [lanes 3] or trypsinized [− tails] [lanes 4]) were separated by SDS-PAGE (16.5%) and stained with Coomassie blue (A) or subjected to immunoblotting, whereupon the membrane was probed with purified CSB (B) or mock incubated (C). Anti-CSB antibodies detected binding of CSB to the immobilized histones (see Materials and Methods for details). No binding of CSB to the tailless histones was detected, suggesting that histone N-terminal tails are important for CSB-core histone interaction. The positions of prestained molecular mass markers (PM) are shown.

FIG. 7
FIG. 7

Remodeling of tailed (+ tails) and tailless (− tails) mononucleosomes by CSB. (A) Reconstituted mononucleosomes were analyzed by electrophoretic mobility shift assay. Nucleosomes with tails are shown (lane 1). Nucleosomes without tails (lane 2) have been generated by trypsinization of labeled tailed nucleosomes after glycerol gradient purification (Materials and Methods). (B) DNase I accessibility assay. Tailed (lanes 2 to 6) and trypsinized (lanes 7 to 11) mononucleosomes were incubated alone (lanes 2 and 7) or with increasing amounts (15, 30 and 60 ng) of CSB in the presence of ATP (lanes 3, 4, and 5, respectively, and 8, 9, and 10, respectively) or with 60 ng of CSB in the absence of ATP (lanes 6 and 11). Remodeling was assessed by DNase I digestion and denaturing gel electrophoresis. Filled arrows, sites of enhanced cutting due to the presence of CSB; open arrows, sites of reduced cleavage. N, naked control DNA (lane 1).

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