The SWI/SNF chromatin-remodeling factor stimulates repair by human excision nuclease in the mononucleosome core particle

Abstract

To investigate the role of chromatin remodeling in nucleotide excision repair, we prepared mononucleosomes with a 200-bp duplex containing an acetylaminofluorene-guanine (AAF-G) adduct at a single site. DNase I footprinting revealed a well-phased nucleosome structure with the AAF-G adduct near the center of twofold symmetry of the nucleosome core. This mononucleosome substrate was used to examine the effect of the SWI/SNF remodeling complex on the activity of human excision nuclease reconstituted from six purified excision repair factors. We found that the three repair factors implicated in damage recognition, RPA, XPA, and XPC, stimulate the remodeling activity of SWI/SNF, which in turn stimulates the removal of the AAF-G adduct from the nucleosome core by the excision nuclease. This is the first demonstration of the stimulation of nucleotide excision repair of a lesion in the nucleosome core by a chromatin-remodeling factor and contrasts with the ACF remodeling factor, which stimulates the removal of lesions from internucleosomal linker regions but not from the nucleosome core.

FIG. 1.

Nucleosome substrate for excision nuclease. The substrate is 200 bp long. (A) Site of AAF-G damage (diamond), restriction sites used for testing remodeling, and approximate location of the area covered by the nucleosome core (ellipse). The substrate was radiolabeled with ³²P at either of two sites (asterisk). The internally labeled substrate was used for excision and remodeling assays, and the terminally labeled duplex was used for footprinting analysis. (B) Sequence with appropriate landmarks. The triangle indicates AAF-G.

FIG. 2.

DNase I footprint of AAF-G mononucleosome. Naked DNA (D) and nucleosome DNA (N) were treated with DNase I for 3 min and separated on a denaturing polyacrylamide gel (5% in 2× TBE) along with size markers (M). The position of AAF-G is indicated by an arrow, and the DNase I-hypersensitive sites in nucleosomal DNA with the 10-nucleotide periodicity are indicated by open circles.

FIG. 3.

Effect of the SWI/SNF remodeling factor on excision nuclease. AAF-G nucleosome (Nuc) or naked DNA was incubated with human excision nuclease for 90 min at 30°C in the absence or presence of 0.48 nM SWI/SNF. The reaction products were analyzed on a denaturing polyacrylamide gel (8% in 2× TBE). The positions of the excision products and the percentage of the damage excised are indicated.

FIG. 4.

Effect of SWI/SNF on the kinetics of excision of AAF-G from the nucleosome core by human excision nuclease. The AAF-G nucleosome was incubated with six-factor reconstituted human excision nuclease in the absence or presence of 0.48 nM SWI/SNF for the indicated times at 30°C. The reaction products were separated on a denaturing polyacrylamide gel (8% in 2× TBE). The level of excision was quantified with PhosphorImager analysis. (A) Autoradiogram of a kinetics experiment. (B) Plot of average data from three independent experiments, including the one shown in panel A. Symbols: •, absence of SWI/SNF; ○, presence of SWI/SNF. The standard errors were less than 15% the value for each data point.

FIG. 5.

Remodeling of the AAF-G nucleosome by SWI/SNF. An internally labeled AAF-G nucleosome was incubated with either EcoRI or PflMI in excision buffer in the absence or presence of 0.48 nM SWI/SNF for 1 h at 30°C. Following deproteinization, the reaction products were separated on a denaturing polyacrylamide gel (5% in 2× TBE). (A) Site of AAF-G damage (diamond), restriction sites for EcoRI and PflMI, location of ³²P label (asterisk), and approximate location of the area covered by the nucleosome core (ellipse). (B) Autoradiogram of a denaturing polyacrylamide gel. Positions of uncut substrate and restriction enzyme digestion products are indicated by arrows. The levels of digestion as percentages of input substrate are given at the bottom of the gel.

FIG. 6.

Effect of damage recognition factors on SWI/SNF remodeling activity. AAF-G (AAF) and control (Unmodified) nucleosomes were incubated for 50 min at 30°C with EcoRI before the addition of 16 pM SWI/SNF at time zero and three repair factors, RPA, XPA, and XPC, as indicated below, and incubation was continued for the indicated time periods. Reaction products were analyzed on denaturing polyacrylamide gels (5% in 2× TBE). The data points are averages of three independent experiments, and the standard errors for each data point were less than 11% the value for each data point. Symbols: •, no SWI/SNF or repair factors; ▵, SWI/SNF but no repair factors; □, repair factors (RPA, XPA, and XPC) but no SWI/SNF; ○, SWI/SNF and repair factors.

FIG. 7.

Model for the role of SWI/SNF in excision repair. Our data suggest that repair factors recruit SWI/SNF to lesion sites to remodel the nucleosome (left arm) but do not exclude the alternative pathway, in which remodeling by SWI/SNF first accelerates the assembly of the repair factors to form PIC1 (right arm). SWI/SNF may contribute to the subsequent steps, PIC2 and PIC3 formation, as well. Circles indicate histones, asterisks indicate DNA damage, and the half arrow indicates repair synthesis. F/1, XPF/ERCC1.