(A) iPOND (immunoprecipitation of biotinylated EdU-DNA) assay of p53, PCNA and ORC2 in HEK293 cells. No click reaction omits biotin and serves as negative control. E, EdU pulse (10 μM, 10 min), E-Thy, EdU pulse followed by thymidine chase (10 μM), E-HU, EdU pulse followed by hydroxyurea (500 μM). Values are relative p53 band intensities normalized to ORC2 band intensities. (B) Schematic and representative image of SIRF (In Situ Interactions at Replication Forks) assay for interactions between protein and nascent DNA in single cells: nascent DNA is pulse-labeled with EdU a protein of interest is crosslinked to the DNA immediately following the EdU pulse. Alternatively, EdU is washed out and cells are incubated with HU (200–400 μM HU) before crosslinking. Proximity ligation assay (PLA) amplification with antibodies against EdU and the protein of interest will result in a signal only if interactions between the nascent DNA and the protein of interest are in close proximity. No signal is produced if the cell has not incorporated EdU. (C) Quantitation of SIRF assay of epigenetic remodeler MLL3 at unchallenged replication forks and (D) at HU stalled replication forks (yellow x) in HAP-1 p53 null and WT cells. (E) Quantitation of SIRF assay of MRE11 at HU stalled replication forks in HAP-1 p53 null and WT cells. Bars represent the mean and the 95% confidence interval. Significance values are derived from student T-test analysis normalized to the respective EdU-PLA intensities (Supplementary file 1).