Fig 2: An Assay to Monitor DNA Synthesis at ALT Telomeres in APBs(A) Experimental scheme for visualizing EdU incorporation in APBs in cells enriched in G2 with thymidine and CDK1i or with CDK1i. G2 cells were labeled with EdU for 1 or 3 h. EdU and PML were detected by immunofluorescence (IF), and telomeres were detected by FISH using the TelC PNA probe (left). EdU incorporation and cell-cycle profiles of the cell populations tested (right).(B) Representative images showing the localization of EdU, PML, and telomeres at G2 U2OS (ALT+) and HeLa1.3 (ALT-) cells. The cells were synchronized in G2 using thymidine and CDK1i.(C) Quantification of telomeres for their presence in APBs and EdU incorporation. The numbers of total, APB+, and EdU+ telomeres were determined in U2OS cells (2,295 telomeres in 42 cells were analyzed).(D) Representative images showing the localization of EdU, PML, and telomeres in PML knockdown and control U2OS cells.(E and F) U2OS cells were treated with control, PML (E), or BLM (F) siRNAs and synchronized in G2 using CDK1i. Approximately 100 cells were divided into five groups (0, 1 or 2, 3 or 4, 5 or 6, and =7) on the basis of the numbers of EdU+ telomeres.(G) Cells were treated with control, POLD3, or POLD4 siRNA and synchronized using thymidine and CDK1i, and 100 cells were divided into five groups on the basis of the numbers of EdU+ APBs.

Fig 3: Abraxas limits mitotic DNA synthesis via RAD52, and POLD3-dependent and RAD51-independent BIR.a Knocking down RAD52 or POLD3 reduced RPA hyperphosphorylation and MUS81 chromatin loading in Abraxas KO cells treated with CPT. Cells transfected with indicated siRNAs were treated with CPT. Chromatin fraction were prepared and used for western blottings. b RPA hyperphosphorylation in Abraxas KO cells treated with CPT is independent of RAD51. c Increased mitotic DNA synthesis in Abraxas KO cells in response to CPT. EdU-click reaction was performed to detect mitotic DNA synthesis. A representative image of EdU foci (left panel) and quantification of the percentage of EdU foci+ cells were shown as mean value ± SD for U2OS (n = 3 independent experiments with total 292 cells), KO (n = 3 independent experiments with total 265 cells) examined (right panel). Two-tailed unpaired Student’s t-test was used for statistical analysis. ***p = 0.0001. d Inhibition of RAD52 reduces MiDAS in Abraxas KO cells treated with CPT. Cells were treated similarly as above. RAD52 inhibitor was added during EdU pulse labeling and quantification of the percentage of EdU foci+ cells were shown as mean value ± SD for WT (n = 4 independent experiment with total 183 cells), KO (n = 3 independent experiment with total 167 cells), WT/RAD52 inhibitor (n = 3 independent experiments with total 121 cells), KO/RAD52 inhibitor (n = 3 independent experiments with total 109 cells) examined. *p = 0.042. e MUS81 depletion inhibits mitotic DNA synthesis in Abraxas KO cells treated with CPT. Cells transfected with indicated siRNAs were incubated with thymidine before treatment of CPT and assay for mitotic DNA synthesis. Percentage of EdU foci+ cells were quantified and shown as mean value ± SD for WT-siCon (n = 9 independent experiments with total 134 cells), KO-siCon (n = 9 independent experiments with total 108 cells), WT-siMUS81 (n = 4 independent experiments with total 172 cells), and KO-siMUS81 (n = 4 independent experiments with total 141 cells). One-way ANOVA Kruskal–Wallis test was used for statistical analysis. *p = 0.0440 and p = 0.0275. See also Supplementary Fig. 6.

Fig 4: The SprT domain is sufficient to suppress the cell-cycle defects, but not the lesion bypass deficiency, in Sprtn knockout cells. (A) Schematic representation of full-length and truncated Spartan proteins used in this study. The truncated Spartan mutant, denoted as SprT, contains the SprT domain but lacks the C-terminal half. SprT, a zinc metalloprotease-like domain; SHP, a p97/VCP-interacting motif; PIP, PCNA-interacting peptide; UBZ4, ubiquitin-binding zinc-finger 4. (B) (Upper panel) Schematic representation of DNA fiber assays. To visualize ongoing replication, mouse embryonic fibroblasts (MEFs) treated with MeOH or 4-OHT for 48 h were sequentially labeled with IdU and CldU with or without UV irradiation (40 J m-2) between the labeling. (Lower panel) DNA fiber assays were performed in SprtnF/F; Cre-ERT2 MEFs expressing full-length human Spartan (FL) or the SprT domain only (amino acids 1–219). Distribution of replication forks at different CldU/IdU ratios is shown. At least 100 fibers were scored for each sample. Horizontal red lines indicate median values. (C) Cell-cycle profiling of SprtnF/F; Cre-ERT2 MEFs expressing full-length human Spartan (FL) or the SprT domain only (amino acids 1–219). Cells were treated with MeOH or 4-OHT for 48 h, stained with PI and analyzed by flow cytometry. (D) Isolation of proteins on nascent DNA (iPOND) assays showing the localization of Spartan at replication forks. 293T cells were pulsed with EdU for 15 min and then chased with thymidine for 10 and 30 min. Eluted proteins were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted for the indicated proteins. PCNA, POLD1 and POLD3 are shown as controls for replisome proteins that are enriched only at replication forks. Histone H2A is shown as a chromatin protein that is not enriched at replication forks. (E) iPOND assays showing that the isolated SprT domain of Spartan can localized at the fork. iPOND was performed in 293T cells expressing full-length human Spartan (FL) or the SprT domain only after pulse labeling with EdU for 20 min. Proteins at EdU-labeled nascent DNA were isolated and assessed using Western blotting. PCNA is shown as a control for replisome protein.

Fig 5: BIR proteins support fork progression. (A) RNF168 expressing MDA-MB-231 cells were treated with the indicated siRNAs then incubated with CldU for 40 min followed by co-incubation with HU and IdU for 24 h. Fiber lengths are presented as the IdU/CldU ratio, black bar indicates median values (numbers below). A combined minimum of 150 replication forks from two biological replicates were measured. ***P < 0.001 (Mann–Whitney) compared to the Scr siRNA treated control. Inset, Western blots showing the effects of two independent siRNAs on protein expression. (B) RNF168 expressing MDA-MB-231 cells treated with PALB2 targeting or scrambled (Scr) siRNA then incubated with CldU for 30 min followed by co-incubation with HU and IdU for 24 h. Fiber lengths are presented as the IdU/CldU ratio, black bar indicates median values (numbers below). A minimum of 150 replication forks were measured for each condition. ***P < 0.001 (Mann–Whitney test) compared to the Scr siRNA treated control. Inset, western blots showing the effects of two independent siRNAs on protein expression. (C) RNF168 expressing MDA-MB-231 cells were treated with the indicated siRNAs and assessed as described in (A). A minimum of 150 replication forks were measured for each condition. ***P < 0.001 (Mann–Whitney test) compared to the Scr siRNA treated control. (D) MDA-MB-231 cells expressing RNF168 were treated with BRCA1 targeted siRNA, then incubated with CldU for 30 min followed by HU, IdU and 50 µM mirin or DMSO. Fiber lengths for CldU tracts are shown in green and IdU tract lengths shown in red, black bar indicates median values. A minimum of 150 tract lengths were measured for each condition. ***P < 0.001 (Mann–Whitney test) DMSO compared to mirin treated cells. (E) MDA-MB-231 cells were transduced to express GFP or POLD3, treated with scrambled (scr) or BRCA1-targeted siRNA, and subject to western blotting for the indicated proteins. DNA fibers were assessed as described in (A). A minimum of 150 replication forks were measured per condition. *P < 0.05, ***P < 0.001 (Mann–Whitney test).