Sybr gold

The purified nucleosome (0.48 μM), containing H3.1 or CENP-A, was incubated with His₆-tagged PR-Set7 (0.49, 0.97, 1.5, and 1.9 μM) in 5.0 µl of reaction solution, containing 10 mM HEPES-KOH (pH 7.8), 18 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.10 mM EDTA, 80 µM of S-adenosylmethionine, 50 mM NaCl, 0.50 mM DTT, and 15% glycerol, at 25 °C for 30 min. After the incubation, the samples were analyzed by 6% native PAGE with 0.2X TBE buffer at 150 V for 60 min. The gels were stained with SYBR-Gold and imaged with an LAS4000 image analyzer (GE Healthcare).

For detection of replication activities, each 10 μl extract sample was incubated with 2 μM of Cy5-dUTP (GE Healthcare) for the appropriate number of times and was diluted with 200 μl of stop buffer (5-mM EDTA, 200-mM NaCl, 0.5% SDS, 20-mM Tris-HCl, pH 8.0) containing 200 μg/ml Proteinase K (Roche) and 10 μg/ml RNaseA (Sigma-Aldrich) and incubated for 2 h at 37 °C. The genomic DNA was purified by phenol/chloroform extraction and ethanol precipitation and electrophoretically separated by 0.8% tris-acetate EDTA (TAE) agarose gel (neutral condition) or 1% alkaline agarose gel (denaturing condition) as described (50 (link)) and stained with SYBR Gold (Invitrogen). The alkaline gel was neutralized with PBS before SYBR Gold staining because it is pH sensitive. The signals of incorporated Cy5 (Replication activity) and SYBR Gold (total genomic DNA) were scanned with Typhoon FLA 9000 Gel Imager (GE Healthcare) and quantified with ImageJ software.

Extension reactions of the fluorescent-labeled 32-mer primer 5′-Cy5-TGCCAAGCTTGCATGCCTGCAGGTCGACTCTA-3′ annealed to the single-stranded circular M13mp18 template (7 nM) were performed in 12.5 µl of 50 mM Tris (pH 8.0), 1 mM dithiothreitol, 50 mM NaCl, 5 mM MgCl₂, and 200 µM each of dNTPs in the presence or absence of PCNA at the indicated concentrations. DNA polymerization was initiated by addition of DNAPs (25 nM) and was conducted at 60 °C for 30 min. Reactions were quenched on ice by addition of one volume of 90% deionized formamide and 20 mM EDTA, before heating at 95 °C for 5 min. Products were resolved on 1% (w/v) alkaline agarose gel. DNA markers (2-Log DNA ladders, New England Biolabs) were loading in 45% deionized formamide and 10 mM EDTA, and run into the same gel at 4 °C for 14 h 30 min at 30 V. After electrophoresis, gels were stained with SYBR Gold (Invitrogen). Gels were first scanned with a Mode Imager Typhoon 9500 (GE Healthcare) for Cy5 to visualize the Cy5-labeled products and then scanned for SYBR Gold for detecting the ladders. Full-length 7249 bp (%) corresponds to the intensity of 7249 bp bands as a percentage of total lane intensity. In all cases, the background value was subtracted. The mean of percentage ± SD of full-length 7249 bp (%) from three independent experiments were obtained (the raw data are provided in Supplementary Table 2).