Micrococcal nuclease mnase

At the designated time points, cells were fixed and crosslinked for 10 min in 1% PBS-buffered formaldehyde. The reaction was stopped by incubation for 5 min with 125mM glycine. Nuclei were isolated (0.3M sucrose, 2mM MgOAc₂, 1% Nonidet P-40, 10mM HEPES (pH7.8)) by centrifugation at 1000 X g for 5 min at 4°C. Bare, unbound genomic DNA and mononucleosomally-protected DNA were isolated from each sample and purified as described previously [12 (link)]. A titration of micrococcal nuclease (MNase; 1–1.25U/mL; Worthington Biochemical Corp.) was used to digest bare, unbound DNA and mononucleosomally-protected DNA [12 (link)].

MEF nuclei from 1 × 10⁷ were isolated using 15 ml of hypotonic buffer A (15 mM HEPES pH 7.9, 10 mM KCl, 5 mM MgCl₂, 0.5 mM EDTA, 1 mM DTT, 0.8 mM PMSF, 1 mM benzamidine, 1 mM sodium metabisulfite) followed by centrifugation at 4000 rpm. Nuclei were resuspended in 6 ml of buffer AC (15 mM HEPES pH 7.9, 30 mM KCl, 3 mM CaCl₂, 1 mM DTT, 0.4 mM PMSF, and 2× protease inhibitor cocktail) and incubated with Micrococcal Nuclease (MNase) (Worthington) at a final concentration of 25 U ml⁻¹ for 20 min. The reaction was stopped by addition of 3 mM EGTA, 5 mM EDTA, 0.1% Triton-X100, and 150 mM KCl (final concentrations). The suspension was centrifuged at 14,000 rpm for 10 min and HA-FLAG-tagged H3.3-containing mononucleosomes were subsequently purified from the supernatant by incubation with 150 µl of M2 anti-FLAG resin (Sigma Aldrich) for 3 h at 4 °C. After washing twice with 150 mM NaCl and once with 300 mM NaCl in wash buffer (20 mM HEPES, 1 mM EDTA, 10% glycerol, 0.01% NP-40, 1 mM BME, 0.4 mM PMSF, 1 mM benzamidine, 1 mM sodium metabisulfite), mononucleosomes were recovered from the beads in elution buffer (wash buffer supplemented with 150 mM NaCl and 500 ng µl⁻¹ 3×FLAG peptide).

pEGFP (500 ng) was digested or not with Nt.BsmA1 (20 U) and then mock-treated or treated with 10 U CIP (New England Biolabs). Following purification (QIAquick spin column; Qiagen), the DNA was incubated with different concentrations of micrococcal nuclease (MNase)(Worthington) in 50 mM Tris-HCl 7.5, 5 mM CaCl₂ and 0.1 mg ml⁻¹ bovine serum albumin for 30 min at room temperature. Reactions were stopped with EDTA (50 mM final) and the DNA purified as above. DNA from the MNase concentration that produced the greatest SSB/DSB ratio (0.015 U) was mock-treated or treated with T4 PNK in the presence of 2 mM ATP and 10U T4 PNK enzyme (wild-type or 3′-phosphatase dead; New England Biolabs). The synthetic oligonucleotide sequences (MWG or Eurogentec) employed to generate duplex substrates are listed in Supplementary Table 1.