Fig. 3: USP1 autocleavage mutant colocalizes strongly with sites of nascent DNA synthesis.
From: USP1-trapping lesions as a source of DNA replication stress and genomic instability
a HCT116 USP1 KO cells complemented with the indicated HaloTag-USP1 fusion constructs were either subjected to immunoblotting analysis (left panel) or DNA fiber analysis of elongating fork speed (right panel). Data are graphed with mean and −/+ SD for 3 independent experiments (n = 200 elongating forks), and p values were calculated using the Mann–Whitney rank-sum t-test (*p < 0.05, ****p < 0.0001, two-tailed). b Cells treated as in (a) were analyzed using the DNA fiber assay for % origin firing. See Supplementary Fig. 2b for criteria for scoring of replication origins. Data for % origin firing are represented by mean and −/+ SD for three independent experiments (n = 200 events) and p-values were calculated using t-test with Welch’s correction (ns = no significance, *p < 0.05, **p < 0.01, two-tailed). c Untreated HCT116 USP1 KO cells expressing the indicated HaloTag-USP1 fusion constructs were labeled with 10 μM EdU and 20 nM JF549 HaloTag ligand for 15 min prior to CSK extraction of soluble proteins and fixation. d–f Representative super-resolution images with EdU signal in magenta are shown. Quantifications of average EdU density per nucleus (d), EdU intensity per focus (e), and number of EdU foci per nucleus (f) are plotted for each sample, based on at least two independent experiments (mean −/+ std, n = 186, 196, 76, 113 nuclei for Mock, USP1-WT, USP1-C90S, and USP1-GG/AA, respectively, p-values were calculated using the student’s t-test; mean and std are represented as the middle bar and the box, respectively). g Representative super-resolution images showing distribution of both the HaloTag-USP1 fusions (green) and EdU-labeled DNA. h Measurements of average USP1 density around EdU are shown, based on at least two independent experiments (mean −/+ std, n = 188, 71, 113 nuclei for USP1-WT, USP1-C90S, and USP1-GG/AA, respectively, p-values were calculated using the student’s t-test; mean and std are represented as the middle bar and the box, respectively).
