Isolation and Analysis of Replication Intermediates

Total genomic DNA was prepared as previously described [21 (link)]. An amount of 200 μg of genomic DNA was digested with restriction enzymes at 37 °C for 4 h, precipitated by ethanol, and resuspended in 400 μL of TNE buffer (100 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 8.0). The digested DNA was then applied to 200 μL of benzoylated naphthoylated DEAE (BND)-cellulose (Sigma-Aldrich). RIs were bound to BND-cellulose, and non-specific binding DNA fragments were washed out with 400 μL of TNE buffer five times. An amount of 200 μL of 1.8% caffeine in TNE was used to elute RIs from BND-cellulose. DNA was then precipitated with isopropanol, washed with 70% ethanol, and resuspended in TE buffer. For B rDNA strains, the enriched RIs were directly applied onto gel electrophoresis. For strains containing C3 rDNA in the developing MAC and B rDNA in the parental MAC, RIs were further digested by SphI to separate parental B rDNA from progeny C3 rDNA 5′ NTS fragments.
Neutral–neutral 2D gel electrophoresis was performed according to a previous description [21 (link)]. Typically, 5–10 μg of BND cellulose-enriched RIs was loaded on a 0.4% agarose gel, and the one-dimensional gel was run in 1×TAE buffer at 1.5 V/cm for 18 h at RT. The gel was visualized by ethidium bromide staining, and a gel slice from each lane was cut with the correct size range of RIs and rotated 90 degrees for two-dimensional gel electrophoresis. Gel slices were inserted into 1% agarose gel, and the two-dimensional gel electrophoresis was performed in 1×TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0) containing 0.5 μg/mL of ethidium bromide at 3 V/cm for 18 h at 4 °C. Southern blot analysis was carried out to detect the patterns of RIs as previously described [21 (link)].