Fig 2: CDT2-dependent down-regulation of SET8 causes nascent strand degradation in DCAF14-deficient cells.(A) U2OS cells transfected with the indicated siRNAs were subjected to fork protection analysis. MG132 was added concomitantly with HU for 4 h where indicated. (B) U2OS cells transfected with the indicated siRNAs were subjected to fork protection analysis. (C) U2OS cells transfected with the indicated siRNAs were subjected to fork elongation assays in the presence of 100 nM CPT as shown. MG132 was added concomitantly with CPT for 30 min where indicated. (D) siNT- and siDCAF14-transfected U2OS cells were pulsed with EdU followed by treatment with HU for 4 h, concomitant with DMSO or p97i as indicated, and subjected to immunofluorescence analyses. Mean nuclei intensity of SET8, CDT1 or p21 was measured by quantitative imaging after preselecting EdU+ nuclei. Graph depicts mean ± SEM using at least 300 nuclei. (E) U2OS cells transfected with the indicated siRNAs were subjected to fork protection analysis. p97i was added concomitantly with HU for 4 h where indicated. (F) Parental and DCAF14 KO U2OS cells transfected with the indicated siRNAs were subjected to fork protection analysis. UNC0379 was added concomitantly with HU for 4 h where indicated. (G) Parental and DCAF14 KO U2OS cells transfected with the indicated siRNAs were subjected to fork protection analysis. MG132 was added concomitantly with HU for 4 h where indicated. Fiber-labeling experiments depict mean ± SEM and at least 100 fibers were quantified.Source data are available for this figure.

Fig 3: RepID depletion confers resistance to the neddylation inhibitor MLN4924. a U2OS WT or RepID KO cells were incubated with or without 250 and 500 nM MLN4924 for 2 days. Cells were labeled with EdU for 30 min prior to collection and analyzed by FACS. Percentage of re-replicating cells is indicated. b Bar chart depicting the cell cycle distribution of cells collected in a. c Chromatin-bound (CB) proteins isolated as in a and analyzed by immunoblotting using antibodies directed against CRL4 substrates including CDT1, SET8, and p21. d WT, RepID KO U2OS cells and RepID KO U2OS cells stably transfected with RepID variants were treated with 500 nM MLN4924 for 2 days and labeled with EdU for 30 min prior to collection and analyzed by FACS. Percentage of subG1 and re-replicating cells are indicated (upper panels). Percentages of chromatin-bound CDT1 in re-replicating cells are indicated (red, bottom panel). e Graphs depicting the percentage of chromatin-bound CDT1 in cells in G2/M phase (left panel) or in re-replicating cells (right panel). The intensity of chromatin-bound CDT1 in specific cell cycle fractionated cells isolated as in d was measured by immunoblotting (bottom panel). The numbers under the panel represent the ratios of CDT1 intensities relative to the intensity of the signal in MLN4924-treated RepID WT cells after normalization with histone H3 from three independent experiments

Fig 4: Model depicting RepID roles in regulating CRL4 activity and RepID-independent SCF functions during replication. In RepID wild-type (RepID WT) cells, CUL4 is recruited to chromatin by interacting with RepID (architectural DCAF) during G1 in the PCNA-free stage. During S-phase, PCNA is loaded and the CRL4-PCNA complex induces CDT1 ubiquitination using CDT2 (functional DCAF) by associating with early replication origins. Additional CUL4 can be recruited by PCNA during the G1/S transition and early S-phase. The CRL4-PCNA complex associates with CDT2 to degrade CDT1 later during S-phase. SCF is also recruited to prevent CDT1 accumulation at late replication origins. In cells expressing RepID, the SKP2 inhibitor has little effect because of the major role of CRL4 in degrading CDT1. In RepID depleted (RepID KO) cells, CUL4 loading to chromatin is compromised because RepID is not available to recruit it to chromatin during the G1 phase. During S-phase, some PCNA-dependent CUL4 is recruited, but CDT1 degradation is reduced because of low chromatin-bound CUL4 levels. Thus, in RepID KO cells, SCF recruitment to late replication origins compensates for the lack of CRL4 and have a greater role in targeting CDT1. Because SCF now acts as the major ubiquitin machinery for CDT1 degradation, the SKP2 inhibitor shows increased potency, and treatment leads to increased CDT1 levels and re-replication-mediated cancer cell death

Fig 5: The expression of Cdc6 and Cdt1 in breast cell lines. Histograms showing (A) Cdc6 and (B) Cdt1 mRNA expression as measured by qPCR in normal breast epithelial cell line MCF10A, and two breast cancer cell lines, MCF7 and MDA MB231 in medium without Fetal Bovine Serum (FBS). (C) Western blot analysis of Cdc6, Cdt1 and β-actin in MCF10A, MCF7 and MDA MB231 cells in normal culture media (left panel) and in media without Fetal Bovine Serum (FBS; right panel).