Fig 2: MiDAS regions are mostly devoid of DNA replication origins.a Top panels, MiDAS signal (purple) and nascent transcription (green, forward transcription; red, reverse transcription) of non-transfected U2OS cells. Bottom panels, origin firing profiles (olive-green) of control (Ctrl) and RIF1-depleted U2OS cells at the EXOC4 locus. For origin firing, the cells were synchronized by mitotic shake-off, released from the mitotic block and allowed to progress through G1 and into S phase over 14 h in the presence of EdU, to label nascent DNA, and hydroxyurea, to limit fork progression. Under these conditions, in control cells, origin firing is observed only in the early S-replicating part of the genome. Bin resolution and ruler scale are as in Fig. 1b. Tx, transcription. b Genome-wide average origin firing signal (green) in control and RIF1-depleted cells at double-peak (left panels) and merged- and single-peak (right panels) MiDAS regions, in comparison to the genome-wide average MiDAS signal (purple). Span of genomic region, 2.9 Mb (double-peak) or 2.3 Mb (merged- and single-peak); Pk, peak.

Fig 3: Histone deacetylase (HDAC) inhibitors suppress IRIF formation of 53BP1 and RIF1. (a) U2OS cells treated with DMSO, MS-275, FK228 or UNC0638 were exposed to IR and immunostained with γH2AX and 53BP1. Right panel: Quantification of the cells displaying more than 20 53BP1 foci. Error bars, SD of two independent experiments, each based on more than 40 cells. Significant differences were calculated compared to DMSO-treated cells: *P = 0.013. (b) U2OS cells treated as in (a) were immunostained with γH2AX and RIF1. Error bars, SD of two independent experiments, each based on more than 120 cells. *P = 0.024, **P = 0.002. (c) Low-magnification views. Nuclei are outlined with dashed lines.

Fig 4: RIF1 ubiquitination by UHRF1 is important for its accumulation at DSB sites.(a). Abrogation of RIF1 ubiquitination. HEK 293T cells stably expressing RIF1 shRNA were reconstituted with the indicated constructs. In vivo ubiquitination assay was performed as described in Methods. (b) UHRF1 ubiquitinates RIF1 in vitro. An in vitro ubiquitination assay was performed using recombinant UHRF1, ubiquitin, UBC13/Mms2 and UBE1 (See Methods). (c) Ubiquitination of RIF1 is required for its recruitment to the DSB sites. HeLa cells stably expressing RIF1 shRNA were reconstituted with the indicated constructs and synchronized. Flag-RIF1 focus formation was then examined following irradiation (5 Gy) as indicated. Upper: Representative micrographs. Lower: quantification of the positive cells (foci>10 per cell) and cell cycle profile. For each sample, 600 randomly selected cells were counted. Data presented as mean±s.d. of three biological triplicates. RIF1 foci Positive cell percentage compared between WT and 9KR group. *P<0.05. (d) Ubiquitination of RIF1 is required for RAD51 accumulation at DSBs. HeLa cells stably expressing RIF1 shRNA were reconstituted with the indicated constructs. Rad51 foci formation was examined 4 h following irradiation (5 Gy). Upper: Representative micrographs; Lower: Quantification of the positive cells (foci>10 per cell). For each sample, 600 randomly selected cells were counted. Data presented as mean±s.d. of three biological triplicates. *P<0.05. (e) HEK 293T cells stably expressing UHRF1 shRNA were transfected with the indicated constructs and synchronized at G1 or S phase, and 53BP1 and RIF1 interaction was examined. (f) HEK 293T cells stably expressing UHRF1 shRNA were reconstituted with the indicated constructs. 53BP1 and RIF1 interaction was examined following synchronization at S phase. (g,h) Ubiquitination of RIF1 is required for its function in DSB repair. RIF1 was depleted in HEK 293 cells integrated with HR or NHEJ reporter. Cells were reconstituted with the indicated constructs and subjected to HR assay (g) and NHEJ assay (h) as described in the Methods. Data presented as mean±s.d. of three biological triplicates. Positive cell percentage compared with control group. *P<0.05. NS: no siginficant difference. (i) Schematic model illustrating Figs 4.

Fig 5: Rescue of 53BP1 IRIF in MG132-treated U2OS cells. (A)U2OS cells transiently expressing the indicated FLAG-tagged H2AX fusion proteins were pretreated with DMSO or MG132 for 1 h, exposed to IR (9 Gy) and 4 h later processed for immunofluorescence. More than one hundred cells with high levels of FLAG signal were scored for 53BP1 IRIF. More than one hundred cells with no FLAG signal (control cells) from the same slides were also scored. The percentages of cells with more than 10 53BP1 foci per cell (mean ± 1 SD) from 3 independent experiments are indicated. ctl, control cells; Scale bar = 20 μm. (B)U2OS cells transiently expressing FLAG-tagged GFP-H2AX were pretreated with MG132 for 1 h, exposed to IR (9 Gy) and 4 h later processed for immunofluorescence for GFP, endogenous 53BP1 and conjugated ubiquitin (FK2). (C)U2OS cells transiently co-expressing a FLAG-tagged ubiq-H2AX fusion protein and a GFP-tagged 53BP1 polypeptide with a wild-type (WT) or mutant (L1622E) RCTD/UDR motif were pretreated with DMSO or MG132 for 1 h, exposed to IR (5 Gy) and 2 h later processed for immunofluorescence with antibodies specific for the FLAG tag and GFP. Scale bar = 10 μm. (D)U2OS cells transiently expressing FLAG-tagged GFP-H2AX were pretreated with DMSO or MG132 for 1 h, exposed to IR (9 Gy) and 4 h later processed for immunofluorescence. More than one hundred cells with high levels of FLAG signal were scored for Rif1 or RAP80 IRIF. More than one hundred cells with no FLAG signal (control cells) from the same slides were also scored. The percentages of cells with more than 10 Rif1 foci per cell or more than 5 RAP80 foci per cell (mean ± 1 SD) from 3 ndependent experiments are indicated. ctl, control cells; Scale bar = 10 μm.