FIG 6.

Localization of 53BP1 to replication forks after replication stress. (A) Scheme of iPOND experiment to measure enrichment of 53BP1 after replication stress. Cells pulsed with 10 μM EdU for 10 min were either harvested to obtain nontreated samples or further pulsed with 2 mM HU for 2 h prior to performing click chemistry to biotinylate DNA regions containing EdU. (B) Results of iPOND analysis of proteins enriched at sites of DNA replication following HU treatment (2 mM for 2 h). Log₂ ratios of peptide tags found in HU-treated versus nontreated samples of 293T cells are shown (n = 2). Error bars show SDs. (C) Confocal microscopy images of RPA, 53BP1, and EdU staining in fixed B cell nuclei without treatment. The merged image shows nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) in blue, RPA in red, and 53BP1 in green. The scale bars represent 4 μm. (D) Confocal microscopy images of RPA, 53BP1, and EdU staining in fixed B cell nuclei after treatment with 4 mM HU for 3 h. The merged image shows nuclear counterstaining with DAPI in blue, RPA in red, and 53BP1 in green. The scale bars represent 4 μm. (E) Quantification of images as shown in panels A and B. Colocalization is shown between RPA and 53BP1 in untreated and HU-treated cells and between RPA and TopBP1 in cells treated with HU. Only EdU⁺ cells were scored. Each dot represents one cell (n = 295 for RPA-53BP1 colocalization measurements in untreated cells, n = 213 for RPA-53BP1 colocalization in HU-treated cells, and n = 156 for RPA-TopBP1 colocalization in HU-treated cells). Red bars indicate medians. The P value was calculated by the two-tailed Mann-Whitney U test. (F) Analysis of iPOND data from WT and 53BP1^−/− B cells after HU treatment (4 mM HU for 3 h). Total MS reads for each protein were corrected using the no-click control and normalized to reads for histone H4 in each sample. The graph shows the ratio of reads in the 53BP1^−/− B cells divided by reads in the WT sample (n = 4). Means ± SDs are shown. *, P value of <0.05, consistent with statistical significance, calculated using Student's t test.