Fig. 4: Spartan facilitates removal of USP1 from forks to enable replication elongation.
From: USP1-trapping lesions as a source of DNA replication stress and genomic instability
a Schematic representation of Spartan-mediated removal of DPCs and tightly bound proteins during DNA replication. b (left) HCT116 cells were treated with either control or SPRTN siRNAs 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 three independent experiments (n = 200 elongating forks) with mean −/+ SD indicated and p values were calculated using the Mann–Whitney rank-sum t-test (ns = no significance, *p < 0.05, **p < 0.01, ****p < 0.0001, two-tailed). (right) Representative tracts of HCT116 USP1-C90S#1 cells treated with either siCtrl or siSPRTN-1. Scale bar = 5 μm. c HCT116 USP1-C90S cells were treated with either siCtrl or siSPRTN-1 siRNAs and complemented with the indicated siRNA-resistant Flag-SPRTN constructs. Following transfections, cells were pulse-labeled with IdU and then CldU for 30 min to assess fork speed of elongating forks. CldU tract lengths are plotted for three independent experiments (n = 200 elongating forks) with mean −/+ SD indicated and p values were calculated using the Mann–Whitney rank-sum t-test (ns = no significance, ****p < 0.0001, two tailed). d A representative maximum intensity projection image of Supplementary Movie 4 showing live-cell imaging of a U2OS cell expressing JF646-Halo-USP1-WT. Brighter spots indicate longer retention. e Representative intensity trajectories of the single USP1 molecules marked in (d). The dwell time (tauon) is labeled in blue. f Normalized distribution of tauon for multiple USP1-WT molecules obtained from N = 16 nuclei. The distribution was fitted with two exponential decays, of which the faster decay corresponds to the average dwell duration of USP1 molecules, while the slower decay provides the photo-bleaching kinetics. g (Left) The average dwell duration of different USP1 molecules as indicated. Mean −/+ std is the fitting result and the std of the fitting of the distribution of tauon built from N = 16, 33, 45, and 33 nuclei for WT, C90S, GG/AA, and Q672A, respectively. (Right) Number of molecules retained longer than 0.6 s in each cell with different USP1 expression as indicated (median: dark bar, box: 25–75% (1st and 3rd quartile), Whisker: minimum and maximum, N = 16, 33, 45, and 33 nuclei for WT, C90S, GG/AA, and Q672A, respectively. P-values were calculated with student’s t-test.
