Fig 2: Delocalized patterns of replication initiation and termination within long genes in USP1 autocleavage mutant cell lines.a (upper panel) Schematic showing anticipated Okazaki fragment distributions around replication origins with strong (blue) versus weak (red) localized firing efficiencies. (lower panel) Schematic representation of Okazaki fragment distributions arising from replication termination at TTS under normal conditions (black) versus delocalized termination under replication stress (red). b Percentage of replication forks moving left to right around TSS binned by total RNA-seq read depth quartile (FPKM), (from ref. 64) for the indicated cell lines. c Percentage of replication forks moving left to right around TSS of actively transcribed genes (FPKM>median), binned by gene length according to quartiles for transcribed genes. d Same analysis as in (c), binned for long genes of 50–100 kb (upper panel) or >100 kb (lower panel). e Percentage of replication forks moving left to right around TTS binned by RNA-seq read depth quartile (from ref. 64) for the indicated cell lines. f Percentage of replication forks moving left to right around TTS of actively transcribed genes (FPKM>median), binned by gene length according to quartiles for transcribed genes. g Same analysis as in (f), binned for long genes of 50–100 kb (upper panel) or >100 kb (lower panel). P values displayed in tables were calculated using the Kruskal–Wallis test, using the regions ±1–10 kb around TSS and TTS of long genes (>100 kb). Effect sizes were calculated using the same regions. The values were calculated comparing the USP1 mutant cell lines against the USP1 wild-type cell line.

Fig 3: USP1 autocleavage mutant colocalizes strongly with sites of nascent DNA synthesis.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).

Fig 4: Spartan facilitates removal of USP1 from forks to enable replication elongation.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.

Fig 5: Model for the role of USP1 activity and/or autocleavage during normal DNA replication progression.During unperturbed replication, low levels of PCNA ubiquitylation allow for efficient TLS to overcome various replicative lesions, and autocleavage allows USP1 to be dynamically recruited to the fork. By contrast, the complete absence of USP1 activity leads to increased aberrant TLS Pol usage due to hyperubiquitylation of PCNA, resulting in reduced replication fork speed and low-level genomic instability. Impaired USP1 autocleavage caused by mutations disrupting the catalytic activity (C90S) or autocleavage site (GG/AA) additionally causes increased fork stalling and replication stress due to increased retention (i.e., “trapping”) of USP1 on DNA. The DNA-metalloprotease SPRTN is efficiently recruited to remove the trapped USP1 catalytic-mutant to mitigate these effects, owing to high Ub-PCNA levels in cells expressing this form of USP1. USP1 hyperactivity and deubiquitylation of PCNA in cells harboring the autocleavage-deficient GG/AA mutant, on the other hand, does not allow for effective SPRTN-mediated removal, resulting in high-level USP1 trapping. Thus, we propose that proper regulation of USP1 activity and autocleavage are both important for ensuring normal USP1 dynamics at the fork and genome stability.