Analyzing DNA Replication Intermediates

In vivo psoralen cross-linking, isolation of genomic DNA from mammalian cells, enrichment of replication intermediates, and analysis of data was performed as previously described (Neelsen et al., 2014 (link); Thangavel et al., 2015 (link)). Briefly, 5–10 × 10⁶ Mouse Embryonic Fibroblasts (Rosa^+/ERCRE Atm^+/C) cells were harvested and genomic DNA was cross-linked by three rounds of incubation in 10 µg/ml 4,5′,8-trimethylpsoralen (Sigma-Aldrich) and 3 min of irradiation with 366 nm UV light on a precooled metal block. Cells were lysed and cellular proteins were digested with 2 mg proteinase K (Life technologies) in 5 ml digestion buffer. DNA was purified by isopropanol precipitation, restriction digested with PvuII HF for 4 hr at 37°C, and replication intermediates were enriched using benzolylated naphthoylated DEAE-cellulose (Sigma–Aldrich) in 3 ml BioRad poly-prep chromatography columns. Samples were prepared by spreading DNA on carbon-coated grids in the presence of benzyl-dimethyl-alkylammonium chloride and formamide. Rotary platinum shadowing was performed using the Balzers BAF400 with Quartz crystal thin film monitor. Images were acquired on a JOEL 1200 EX transmission electron microscope with side-mounted camera (AMTXR41 supported by AMT software v601) and analyzed with ImageJ (National Institutes of Health).