Benzonase

The chromatin-derived fraction was separated as previously reported [81 (link), 82 (link)]. First, cells were lysed with buffer A (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES (pH 6.8), 1 mM EGTA, and 0.2% Triton X-100; containing protease and phosphatase inhibitors) for 30 min on ice. Then, the chromatin-containing pellet was separated from the soluble fraction by centrifuging crude lysates at 14,000 rpm at 4 °C for 10 min. The pellet was digested with RIPA buffer containing phosphatase inhibitor, protease inhibitor and 125 U of benzonase (Yeasen, Shanghai, China) for another 40 min to acquire chromatin-bound proteins. The chromatin-containing supernatants were clarified by centrifugation at 14,000 rpm and 4 °C for 10 min to remove debris, and the protein concentration was quantified by a Pierce BCA protein assay kit (Thermo Scientific™).

Yeast cells culture (A600 ≈ 1.0) with or without MMS treatment were collected and lysed on a bead beater in lysis buffer (100 × 10⁻³m HEPES, pH 8.0, 20 × 10⁻³m MgCl₂, 150 × 10⁻³m NaCl, 10% glycerol, 0.4% Nonidet P‐40, 0.1 × 10⁻³m EDTA plus protease and phosphatase inhibitors). 500U mL⁻¹ of benzonase (YEASEN) was added to each sample prior to cell lysis. The extract was clarified by centrifugation at 12,000 g for 10 min at 4 °C, followed by incubating with protein G‐agarose beads for 1 h at 4 °C to preclear non‐specific binding. After centrifugation, the supernatant was incubated with anti‐HA(MBL) or anti‐FLAG (Sigma) antibody at 4 °C overnight with agitation. Protein G‐agarose beads were added, and the mixtures were incubated for another 3 h at 4 °C. Subsequently, the beads were subjected to extensive washing with the lysis buffer at 4 °C. Immunoprecipitated proteins were eluted by boiling beads in a 2xSDS loading buffer for 5 min.

The procedure of protein aggregates was prepared according to a previously reported protocol [65 (link)]. 50 OD₆₀₀ units of 12 h-inductional strains in BMMY were harvested by centrifugation (3000g) for 3 min at 4 °C, and pellets were flash frozen in liquid nitrogen. Pellets were washed with 20 mM potassium phosphate (pH 6.8) and resuspended in buffer II [20 mM potassium phosphate pH 6.8, 1 mM EDTA, 10 mM DTT, 0.1% Tween 20, protease inhibitor cocktail (MedChemExpress), 1 mM PMSF (Yeasen, shanghai, China), 150 U ml⁻¹ lyticase and 1.25 U ml⁻¹ Benzonase (Yeasen, shanghai, China)], incubated for 15 min at 30 °C and chilled on ice for 5 min. After sonication, the samples were collected by centrifugation (200g) for 20 min. The supernatants were diluted to identical protein concentrations and taken as input control (total). Aggregates were sedimented by centrifugation (16,000g) for 20 min at 4 °C. The aggregated proteins were washed twice with buffer II (20 mM potassium phosphate pH 6.8, 2% (v/v) NP-40, 1 mM PMSF, protease inhibitor cocktail), sonicated and centrifuged at 16,000g at 4 °C for 20 min. Protein aggregates were sonicated with rehydration buffer (7 M urea, 2% CHAPS, 2 M thiourea, 20 mM DTT, 1% SDS) [36 (link)], boiled in SDS sample buffer, together with totals separated by 12% SDS-PAGE, and analyzed by Coomassie blue staining and western blotting.