Nuclease p1

o-Toluidine hydrochloride (T536215; purity, 97%) was obtained from Toronto Research Chemicals, Toronto, ON, Canada, p-toluidine (PT) hydrochloride (T0303; purity, >98%) was obtained from Tokyo Chemical Industry, Tokyo, Japan, and aniline (ANL) hydrochloride (017-04092; purity, 97%) was obtained from Wako Pure Chemical Industries, Osaka, Japan. AAOT (A1020; purity, >98%) and apocynin (H0261; purity, >98%) were obtained from the Tokyo Chemical Industry. Nuclease P1 and HPLC-grade acetonitrile were purchased from Wako Pure Chemical Industries, Ltd. Phosphodiesterase I was purchased from Worthington Biochem (Lakewood, NJ, USA). Bovine spleen Phosphodiesterase II, DNase I, and bacterial alkaline phosphatase Type III (Escherichia coli) were purchased from SigmaAldrich (St. Louis, MO, USA). All other chemicals used were of analytical grade and purchased from Wako Pure Chemical Industries, Ltd.

The internally labeled tRNA transcript (containing ³²P at around 3000 cpm) and 150 ng of the purified cmPus1 or cmPus4 in 50-µL buffer D (100 mM Tris-HCl (pH 8.0), 100 mM ammonium acetate, 5 mM MgCl₂, 2 mM DTT, 0.1 mM EDTA) were incubated at 37 °C for 1 or 2 h. The tRNA was extracted with phenol-chloroform and then recovered by ethanol precipitation. 0.8 A₂₆₀units total RNA from Lactobacillus plantarum was added before ethanol precipitation. The tRNA pellet was dissolved and digested overnight at 37 °C with 3 µL (1 unit) nuclease P1 (Wako, Tokyo, Japan) in 50 mM ammonium acetate (pH 5.0) or RNase T2 in 20 mM ammonium acetate (pH 5.0). nuclease P1 or RNase T2 was used for UTP- or ATP- and CTP-labeled tRNA transcripts, respectively. An aliquot of the sample was spotted onto a thin layer plate (Merck, Darmstadt, Germany, TLC Cellulose F; 10 cm × 10 cm) and separated as described previously [47 (link)]. The ³²P-labeled nucleotides were monitored using a Typhoon FLA 7000 imager (GE Healthcare, United States). Standard nucleotides were marked by UV_260nm irradiation.

The measurement of global DNA methylation by HPLC was performed essentially as described35 (link). In brief, genomic DNA was prepared from MEFs or various mouse tissues. The RNA-free genomic DNA samples were treated with nuclease P1 (Wako) and cloned alkaline phosphatase (CIAP, NEB) before they were subjected to HPLC (Agilent 2000 Series, Agilent Eclipase XDB-C18) analysis.

DNA associated with Cut14-PK, histone H3 or Rad21-GFP was purified using the ChIP procedure as described. Before elution from antibody-bound magnetic beads, DNA on beads was treated with nuclease under the following conditions. All of the reactions were performed in a volume of 100 μl using input DNA derived from 1.5 × 10⁸ yeast cells. For nuclease P1 (Wako Pure Chemical Industries) treatment, 10 U enzyme was used in a buffer comprising 40 mM MES-Na, 400 mM NaCl, 0.2 × cOmplete-EDTA protease inhibitor cocktail (Roche), 50 μg ml⁻¹ BSA and 1 μg ml⁻¹ fragmented λ-DNA at pH 6.0 and incubated for 20 min at 42 °C. For RNase A (Roche) treatment, 50 μg enzyme was used in a buffer containing 10 mM Tris-Cl, 1 mM EDTA and 0.2 × cOmplete-EDTA protease inhibitor cocktail at pH 8.0, and incubated for 20 min at 37 °C. For RNase H (Takara Bio) treatment, 60 U enzyme was used in a buffer containing 40 mM Tris-Cl, 4 mM MgCl₂, 0.2 × cOmplete-EDTA protease inhibitor cocktail and 50 μg ml⁻¹ BSA at pH 8.0 and incubated for 20 min at 37 °C. On completion of the reactions, beads were collected and washed once with TE buffer and then DNA was eluted, reverse-crosslinked and purified as described21 (link). For analysis of DNA binding to human NCAPG, DNA purified from 5 × 10⁶ cells was processed as described above.

PhIP-HCl was purchased from the Nard Institute (Osaka, Japan), and its purity was confirmed to be >99% by HPLC. AA was obtained from Sigma Aldrich (St. Louis, MO, United States). Nuclease P1 was purchased from Wako Pure Chemical Industries (Osaka, Japan). Phosphodiesterase I was purchased from Worthington. Nicotinamide, bovine spleen Phosphodiesterase II, DNase I, and bacterial alkaline phosphatase type III (E. coli) were purchased from Sigma Aldrich (St. Louis, MO, United States). Difco™ Nutrient Broth and Cofactor-I were acquired from Becton, Dickinson and Company (Sparks, MD, United States), and Oriental Yeast Co., Ltd (Tokyo, Japan), respectively. The S9 mix was purchased from the IEDA Trading Corporation (Tokyo, Japan). All other chemicals used in the study were of analytical grade and were purchased from Wako Pure Chemical Industries.