Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription.
Bottom Line: Chromatin compaction of deoxyribonucleic acid (DNA) presents a major challenge to the detection and removal of DNA damage.Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription.Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA.
Affiliation: Department of Genetics, Medical Genetics Cluster, Cancer Genomics Netherlands, Erasmus MC, Rotterdam, 3015 GE, The Netherlands.Show MeSH
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Mentions: CSB (Figure 5C), ACF1 and WSTF (Supplementary Figure S5A and B) were immunoprecipitated using chromatin-enriched nuclear extracts from 10 14-cm culture dishes of GFP-CSB expressing CS1AN cells or five 14-cm culture dishes of U2OS cells expressing ACF1-GFP or GFP-WSTF. Cells were collected 20 min (CSB) or 5 min (ACF1/WSTF) after irradiation (20 J/m2) by scraping in 3 ml of phosphate buffered saline (PBS) containing protease inhibitor cocktail (Roche), centrifuged for 5 min at 1500 rpm and washed again with PBS. Cells were swollen in 5 x pellet volume of Hepes buffer (10-mM HEPES pH 7.6, 1.5-mM MgCl2, 10-mM KCl, 0.5-mM Dithiothreitol, protease inhibitor cocktail) for 10 min. Nuclei were isolated by douncing cells with a type A pestle and centrifugation at 3000 rpm for 10 min at 4°C. Nuclei were washed and resuspended in 1.5 x pellet volumes of Hepes buffer (100-mM HEPES pH 7.6, 1.5-mM MgCl2, 150-mM NaCl, 25% glycerol, protease inhibitor, 0.5-mM Dithiothreitol) and subsequently dounced using a pestle type B. Next, chromatin was digested with 25-U Microccocal nuclease (MNase; Sigma) for 1 h at 4°C. These conditions were chosen such that DNA was digested to mononucleosome size. The resulting chromatin-enriched nucleoplasmic fraction was cleared from insoluble nuclear material by centrifugation at 15 000 rpm for 15 min. For immunoprecipitation of SMARCA5 mutants (Figure 7C), extracts were prepared by scraping cells from a 14-cm dish in Radioimmunoprecipitation assay buffer (PBS containing 1% NP-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulphate; Roche protease inhibitor cocktail) followed by sonication (to obtain DNA fragments <800 bp) and 16 000 g centrifugation at 4°C for 10 min to remove insoluble material. Extracts were incubated with GFP-trap beads (Chromotek) for 2 h at 4°C. Subsequently, beads were washed four times in Hepes buffer and boiled in Laemmli sample buffer for analysis by western blotting.
Affiliation: Department of Genetics, Medical Genetics Cluster, Cancer Genomics Netherlands, Erasmus MC, Rotterdam, 3015 GE, The Netherlands.