Fig 2: 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 [a-32P]dATP, p27kip1 and 50 µM PHA-767491 ± 5 mM caffeine to match the first incubation. At the indicated times, incorporation of [a-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 3: The CDC7i test determines that low FR is the primary effect of PrimPol down-regulation. A, schematic of the simultaneous reduction in FR and IOD (higher origin density) caused by PrimPol down-regulation in HeLa cells (data shown in C–H). The CDC7i test can be applied. B, immunoblots showing the down-regulation of PrimPol and the effect of CDC7i on MCM2 phosphorylation at Ser-53. MEK2 levels are shown as a loading control. The first two lanes show serial 2-fold dilutions of the control condition (third lane) for comparison purposes. C–E, the result of applying the APH test. HeLa cells were treated with doxycycline for 4 days to induce shPrimPol, and 5 µm APH was added for 2 h when indicated. C, distribution of FR values (from left to right; n = 617, 615, 617, and 627). D, origin activity as determined by percentage of first-label origin structures (left panel; n = 1037, 1037, 1013, and 1044 total structures) and distribution of IOD values (right panel; n = 102, 107, 105, and 103). E, representative images of DNA fibers in HeLa cells treated as in C and D. Scale bar = 10 µm. F–H, HeLa cells were treated with doxycycline for 4 days to induce shPrimPol, and 60 µm CDC7i was added for 12 h when indicated. F, distribution of FR values (from left to right, n = 934, 979, 901, and 914). G, origin activity determined by percentage of first-label origin structures (left panel; n = 1514, 1588, 1641, and 1525 total structures) and distribution of IOD values (right panel; n = 141, 157, 37, and 39). H, representative images of DNA fibers in HeLa cells treated as indicated in F and G. Scale bar = 10 µm. In the dot plots, median values are indicated by horizontal lines. Bar graphs represent mean ± S.D. In C–E, data were pooled from two independent experiments. In F–H, data were pooled from three independent experiments.

Fig 4: Human Mcm4 Phosphorylation Predicts DNA Replication(A and B) HeLa cells were synchronized in early M phase by mitotic shakeoff and then replated for different times. (A) DNA content of cells as determined by flow cytometry. (B) Immunoblot of Mcm4, total Mcm2, and Mcm2-P-S40 on and off chromatin. Lamin B and tubulin serve as loading and fractionation controls.(C) Cells were synchronized as in (A) and were replated with 10 µM PHA-767491 or 10 µM XL413 in the media. At 7.5 hr, cells were pulsed for 30 min with EdU and then analyzed by flow cytometry for DNA content and EdU incorporation. An example plot and gate are shown plus the mean and SD of EdU+ cells from three experiments.(D) Cells were synchronized as in (A) and replated for 8 hr (t-2); media was then optionally supplemented with 10 µM PHA-767491 (PHA), 10 µM XL413 or 225 nM tautomycetin (Tauto) as indicated for 2 hr. Cells were then immunoblotted for Mcm4, Mcm2-P-S40, Mcm2-P-S53, or total Mcm2 on and off chromatin. Lamin B and tubulin served as loading and fractionation controls.See also Figure S1.

Fig 5: Phosphorylation of Different MCM Subunits by Xenopus DDK Is Reversed by PP1(A–C) Xenopus egg extracts were supplemented with demembranated sperm nuclei and optionally supplemented with 50 µM PHA-767491 (PHA) or 100 µM XL413 (XL). (A) After incubation for 40 min, isolated chromatin along with 0.5% (v/v) of the supernatant was immunoblotted for Mcm4, Mcm2-P-S40, Mcm2-P-S53, and total Mcm2. (B) At the indicated times, chromatin was isolated and immunoblotted for Mcm4, Mcm2-P-S40, Mcm2-P-S53, total Mcm2, PCNA, and histone H3. (C) Extract was supplemented with [a-32P]dATP. Total DNA synthesis was determined at indicated times.(D and E) Sperm nuclei were incubated for 60 min in Xenopus egg extracts treated with p27kip1 to allow Mcm4 hyperphosphorylation. 50 µM PHA-767491 or 100 µM XL413 plus or minus 1 µM tautomycetin (Tauto) was then added, and chromatin was isolated either immediately after inhibitor addition (60 min) or later at every 5 for next 20 min (65–80 min). Chromatin samples were immunoblotted for Mcm4, Mcm2-P-S40, Mcm2-P-S53, total Mcm2, and histone H3.