Fig 2: PIF1 is required for BIR at Flex1 upon replication and oncogenic stress Schematic drawing of the EGFP-Flex1-BIR-5086 reporter. Flex1: 0.3 kb Flex1(AT)34 derived from CFS FRA16D.U2OS (EGFP-Flex1-BIR-5086) cells were treated with or without 2 mM HU for 24 h, and the percentage of EGFP-positive cells was quantified by FACS analysis 3 days after HU removal.U2OS (EGFP-Flex1-BIR-5086) cells expressing shRNAs for RAD51, POLD3, and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 h. The percentage of EGFP-positive cells by HU induction was quantified by FACS analysis 3 days after HU removal.U2OS (EGFP-Flex1-BIR-5086) cells were infected by retrovirus expressing H-RAS-V12-FLAG (RAS) or Cyclin E-HA (Cyc E), and the percentage of EGFP-positive cells was quantified by FACS analysis 4 days after retrovirus infection (top). The expression of RAS or Cyclin E is shown by Western blot analysis (bottom).U2OS (EGFP-Flex1-BIR-5086) cells expressing shRNAs for RAD51, POLD3, and PIF1 or shRNA vector (Ctrl) were infected by retroviruses expressing RAS, and the percentage of EGFP-positive cells was quantified by FACS analysis 4 days after retrovirus infection (top). Expression level of RAD51 and POLD3 is shown by Western blot analysis (bottom).U2OS WT or PIF1-KO cells were infected by retrovirus expressing RAS or empty vector. Cell profiling was plotted by counting cell numbers every 24 h, and normalized to the cell number on the first day. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01. Source data are available online for this figure.

Fig 3: PIF1- and POLD3-mediated BIR is important for repairing DSBs at Flex1 caused by FANCM deficiency U2OS (EGFP-Flex1-BIR-5086) cells were infected by lentiviruses expressing FANCM shRNA or shRNA vector (Ctrl; left) or cells from the same reporter cell line expressing shRNAs for RAD51, POLD3, and PIF1 or shRNA vector (Ctrl) were infected by FANCM shRNA lentiviral viruses (right). The percentage of EGFP-positive cells after spontaneous recombination was quantified by FACS analysis 5 days after lentiviral infection of FANCM shRNA. Knockdown of FANCM by shRNA is shown by qPCR and Western blot in Appendix Fig S12A.U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3, and PIF1 or shRNA vector were infected by lentivirus expressing FANCM shRNA. The percentage of EGFP-positive cells was quantified by FACS analysis 5 days after FANCM shRNA lentiviral infection. Knockdown of FANCM by shRNA is shown by qPCR and Western blot in Appendix Fig S12B.Proposed model for concerted roles of FANCM-dependent fork reversal and PIF1/POLD3-dependent BIR in protection of Flex1 stability. Pink arrow: endonuclease cleavage to remove Flex1.Growth curves of WT or FANCM-KO HCT116 cells were plotted after expressing POLD3 (left) or PIF1 (right) shRNA or shRNA vector (Ctrl). Cell number was normalized to that on day one. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.

Fig 4: BIR is used at replication-associated DSBs even for STGC Proposed models for repair of DSBs generated at broken replication forks (see text for details). seDSB: single-ended DSB; deDSB: double-ended DSB. Pink arrows: endonucleases to remove Flex1 or other secondary structures at DSB ends or DNA tails at the fork junctions.Schematic drawing of the EGFP-STGC-1731 reporter and proposed pathways to repair DSBs generated by endonuclease cleavage (I-SceI or Cas9, left) or converted from nicks (Cas9n, right).U2OS (EGFP-STGC-1731) cell lines carrying Dox-inducible Cas9/sgRNA-1731 (Dox-Cas9) or Cas9n/sgRNA (Dox-Cas9n) were incubated with or without Dox (5 µg/ml), and the percentage of EGFP-positive cells was quantified by FACS analysis 2 days later (top). U2OS (EGFP-STGC-1731, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs for RAD51, POLD3, and PIF1 or shRNA control (Ctrl) were incubated with Dox (5 µg/ml), and the percentage of EGFP-positive cells was quantified by FACS analysis after 2 days (bottom).Schematic drawing of the EGFP-Flex1-STGC-1541 reporter and the proposed mechanism to repair DSBs at Flex1 generated upon fork breakage by SDSA which involves two DSB ends.U2OS (EGFP-Flex1-STGC-1541) cells were treated with or without 2 mM HU for 24 h, and the percentage of EGFP-positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3, and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 h, and the percentage of EGFP-positive cells was quantified by FACS analysis 4 days later (right).U2OS (EGFP-Flex1-STGC-1541) cells were infected by retroviruses expressing RAS or empty vector, and the percentage of EGFP-positive cells was assayed by FACS 5 days following infection (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3, and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS, and the percentage of EGFP-positive cells was assayed by FACS 5 days after infection (middle). Expression level of RAD51 and POLD3 is shown by Western blot analysis (right).Proposed models for activating BIR at replication-dependent and replication-independent DSBs (see text for details). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P > 0.05. Source data are available online for this figure.

Fig 5: POT1 dysfunction triggers mitotic DNA synthesis (MiDAS) at telomeres. (A,B) Validation of growth defects upon inhibition of MiDAS factors, SMC2 and POLD3, in cells expressing mutant POT1. (A) Western blot analysis for POLD3 and SMC2 in cells with the indicated treatment. (B) Graph depicting cellular growth monitored in real time by Incucyte in the indicated cells. (C) Analysis of MiDAS in asynchronous HT1080 cells expressing POT1-WT and POT1-?OB. Quantification of the number of metaphases with EdU foci at telomeres from asynchronous cells. Mean ± SD of three independent experiments (n = 634 metaphases for POT1-WT and 597 for POT1-?OB): Student's t-test, paired, and one-tailed. (D) Setup of MiDAS experiment in RPE-1 p53-/- cells expressing POT1-WT and POT1-?OB. Cells were treated with aphidicolin (0.4 µM) for 40 h and with Cdk1 inhibitor RO-3306 (9 µM) during the last 16h of the aphidicolin treatment. Following release from the G2/M block, cells were incubated with 20 µM EdU and Colcemid for 50–60 min, before metaphases were harvested. (E,F) Analysis of mitotic DNA synthesis (MiDAS) in RPE-1 p53-/- cells exogenously expressing POT1-WT and POT1-?OB following the treatment as in D. (E) Representative image of EdU incorporation at telomeres; arrowheads point to EdU foci at telomeres. (F) Quantification of the number of metaphases with EdU incorporation at telomeres; two independent experiments. (G,H) Analysis of ultrafine DNA bridges (UFBs) in U2OS cells exogenously expressing POT1-WT and POT1-?OB. (G) Representative images of mitotic UFBs detected by indirect immunofluorescence for PICH (green); DNA is stained with DAPI in blue. (H) Quantification of PICH UFBs in two independent experiments (total n > 100 mitoses for each condition).