Fig 2: RIF1 does not control Cdt1 and Cdc6 RIF1 does not control the cell cycle‐specific localization of Cdc6 protein. Subcellular localization of Cdc6 protein was previously shown to be cell cycle regulated through its phosphorylation by CDK, which leads to export of Cdc6 protein from the nucleus to the cytoplasm 47. To test whether Rif1 removal affects Cdc6 regulation, subcellular localization of Cdc6 was analyzed by immunofluorescence microscopy in U2OS cells treated with either siCtrl or siRIF1. Abundance of cytoplasmic Cdc6 and DNA content was measured for more than 1,700 cells under each condition using CellProfiler software as follows. Adaptive thresholding was applied to the blue (DAPI‐stained nuclear DNA) and green (anti‐Cdc6 stain) channels to identify areas corresponding to individual nuclei and cells, respectively. The cytoplasmic area was then defined by excluding the nuclear area from the cell area. For each cell, the integrated intensity in the green (Cdc6) channel was then measured separately for the cytoplasmic area, the nuclear area, and the whole cell. Integrated intensity in the blue channel was measured for each nuclear area to obtain a cellular DNA content value. Cells at the edge of images were excluded. Scatter plots show cytoplasmic Cdc6 abundance (y‐axis; log10 scale) against DNA content (x‐axis), while the histograms (lower panels) show DNA content of the corresponding cells. Representative images are presented at the top. The scale bar indicates 30 μm.RIF1 has only a minor effect on Cdt1 stability. Degradation of Cdt1 protein is regulated in a similar way to ORC1: outside G1 phase, Cdt1 is phosphorylated and degraded 48. The effect of depletion followed by ectopic expression of RIF1 on Cdt1 abundance was tested by flow cytometry. Flp‐In T‐REx 293 cells with stable GFP, GFP‐RIF1, or GFP‐RIF1‐pp1bs constructs were depleted for endogenous RIF1 by siRNA, and 1 day later GFP, GFP‐RIF1, or GFP‐RIF1‐pp1bs was induced. After further 3 days, cells were prepared for flow cytometry and abundance of Cdt1 protein per cell was analyzed by flow cytometry.

Fig 3: RIF1 interacts with protein phosphatase 1 isoforms Construction of RIF1 cDNA mutated at its PP1 interaction motifs. Critical residues in all three potential PP1 interaction motifs are substituted with alanine, to create a RIF‐pp1bs allele.Expression and localization of GFP‐RIF1 fusion proteins in stably transfected cells. Flp‐In T‐REx 293 cells with GFP, GFP‐RIF1, or GFP‐RIF1‐pp1bs were cultivated with 1 μM doxycycline (DOX) for 3 days, and expression and localization of GFP proteins were confirmed by fluorescence microscopy. Phase‐contrast, DAPI‐stain, and GFP images are shown. Scale bar indicates 25 μm.RIF1 binds PP1 protein isoforms through its PP1 interaction motifs. GFP, GFP‐RIF1, and GFP‐RIF1‐pp1bs proteins were recovered from cell extracts using GFP‐Trap beads, and co‐purifying proteins were analyzed by Western blotting with anti‐GFP (upper two panels) or isoform‐specific PP1 antibodies (lower panels).

Fig 4: Rif1-PP1 Regulates Replisome Stability and S-phase Progression(A) Sperm nuclei were incubated for 60 min in Xenopus egg extracts supplemented with 100 μM aphidicolin and 5 mM caffeine. 50 μM PHA-767491 (PHA) ± 1 μM tautomycetin (Tauto) was added for a further 25 min. Chromatin-bound proteins were immunoprecipitated with antibodies against Cdc45 or pre-immune sheep IgG. Samples were immunoblotted for Cdc45, Mcm4, Mcm2-P-S40, Mcm2-P-S53, total Mcm2, and Psf2.(B) Xenopus egg extracts were supplemented with demembranated sperm nuclei and 100 μM aphidicolin. After 60 min, p27kip1 was added and aliquots were supplemented with 50 μM PHA-767491 ± 5 mM caffeine (CAFF). After a further 25 min, chromatin was transferred to extract supplemented with [α-32P]dATP, p27kip1 and 50 μM PHA-767491 ± 5 mM caffeine to match the first incubation. At the indicated times, incorporation of [α-32P]dATP into nascent DNA was determined and expressed as normalized values against the incorporation at 1 min.(C) HeLa cells were treated with control or Rif1 siRNA, synchronized by double thymidine block, and released 2 hr before either treatment with hydroxyurea (HU; 5 mM), aphidicolin (APH; 1 μg/mL), methyl methanesulfonate (MMS; 0.02%), camptothecin (CPT; 5 μM), etoposide (ETO; 5 μM), or UV (∼45 J/m2). EdU was added in the last 30 min of treatment, and 2 hr after the start of the treatments, cells were harvested, fixed, and analyzed by flow cytometry.(D) Model for the role of Rif1 and Cdc7 in controlling replication initiation and replisome stability. Replication initiation is regulated by the phosphorylation of Mcm2-7 by Cdc7, opposed by Rif1/PP1. Active replisomes are also dephosphorylated by Rif1/PP1, which requires ATR/Chk1 to stabilize them in response to replicative stresses.See also Figure S6.

Fig 5: Model of DNA replication control by RIF1‐PP1In normal cells (left panel), RIF1 acts with PP1 to stabilize ORC1, promoting its loading at potential DNA replication origin sites at M/G1 phase (top). ORC1 then stimulates MCM loading, to license origins in G1 phase (middle). When cells enter S phase, RIF1 counteracts DDK‐mediated phosphorylation of the MCM complex to limit the number of origins activated (bottom). In the absence of RIF1 (right panel), ORC1 is short‐lived and its chromatin association reduced (top), so that fewer origins are licensed in G1 (middle). During S phase, however, increased MCM phosphorylation means that a larger fraction of licensed origins are activated (bottom). For simplicity, the ORC2–5 subunits (which are chromatin‐associated throughout the cell cycle) are omitted.