Fig. 1: Impaired USP1 autocleavage contributes to reduced replication fork speed and fork stalling.
From: USP1-trapping lesions as a source of DNA replication stress and genomic instability
a Schematic representations of USP1 mutants utilized in this study. b Whole-cell lysates of HCT116 USP1 mutant cell lines were analyzed by immunoblotting with the indicated antibodies. For this figure and all subsequent figures, tubulin serves as a loading control. Iso. USP1 represents an isogenic wild-type control derived following clonal selection of USP1 mutant cell lines. c (left panel) Representative images of elongating DNA fiber tracts from HCT116 parental cell line and USP1 knock-in clones. Scale bar = 5 μm. (right panel) Scatter plots show CldU tract length measurements from elongating forks for three independent experiments (n = 200 elongating forks) with mean and −/+ SD indicated. p values were calculated using the Mann–Whitney rank-sum t-test (ns = no significance, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-tailed). d HCT116 parental and USP1 mutant cell lines were treated with either control or Polκ siRNA for 72 h, and subsequently pulse-labeled with IdU and then CldU for 20 min. CldU tract lengths were measured in each sample to assess relative speed of elongating forks. Data are plotted for 3 independent experiments (n = 200 elongating forks) with mean and −/+ SD indicated and p values were calculated using the Mann–Whitney rank-sum t-test (ns = no significance, **** = p < 0.0001, two tailed). e Schematic for assessment of symmetrical vs. asymmetrical fork progression in HCT116 cell lines transfected with either control or Polκ siRNAs, as in (d). Representative images show bidirectional forks emanating from a single origin for the indicated cell lines. Scale bar = 5 μm. Scatter plots depict ratios of CldU tract length measurements from leftward- versus rightward-moving forks emanating from the same origin of replication (n = 100 bi-directional forks (origins)), with mean and −/+ SD indicated. p values were calculated using the Mann–Whitney rank-sum t-test (ns = no significance, ****p < 0.0001, two-tailed).
