Poh WT , Chadha GS , Gillespie PJ , Kaldis P , Blow JJ .

083524/Z/07/Z Wellcome Trust , 097945/B/11/Z Wellcome Trust , 14301 Cancer Research UK, BB/H013024/1 Biotechnology and Biological Sciences Research Council , 097945 Wellcome Trust , WT097945 Wellcome Trust

	Figure 1. PHA-767491 inhibits DNA replication and Mcm4 hyperphosphorylation. (a) Interphase Xenopus egg extract was supplemented with demembranated sperm nuclei and [α-32P]dATP plus different concentrations of PHA-767491; after 90 min total DNA synthesis was determined. Mean and s.e.m. of 20 independent experiments is shown. (b) Extracts supplemented with demembranated sperm nuclei plus (ii,iv) or minus (i,iii) 50 μM PHA-767491 were incubated for 40 min; nuclei were then stained with Hoechst 33258 and visualized with phase contrast (iii,iv) or fluorescence microscopy (i,ii). Scale bar, 10 μm. (c,d) Extracts were supplemented with demembranated sperm nuclei plus or minus 50 μM PHA-767491, p27kip1 or geminin. After incubation for the indicated times, chromatin was isolated and immunoblotted for Mcm4, Cdc45, Cdc7, Cdc6 and PCNA. The lower portion of the gel was stained with Coomassie to visualize histones. (e) Egg extract was supplemented with demembranated sperm nuclei and [α-32P]dATP. After incubation for 40 min, aliquots were optionally supplemented with 50 μM PHA-767491, p27kip1 or both. At the indicated times, total DNA synthesis was determined. (f) Egg extract was first incubated with demembranated sperm nuclei for 40 min, and the extract was supplemented with 50 μM PHA-767491. At the indicated times, chromatin was isolated and immunoblotted for Mcm4, Psf2 and PCNA.
	Figure 2. Fork rate and the reversal of Mcm4 hyperphosphorylation by PP1. (a–c) Sperm nuclei were incubated in egg extract supplemented with 100 µM aphidicolin. After 60 min incubation, one aliquot was supplemented with p27kip1 and one aliquot was supplemented with p27kip1 plus 50 μM PHA-767491. After a further 15 min incubation chromatin was isolated. Chromatin was then incubated in extract supplemented with [α-32P]dATP and p27kip1 and optionally with 50 μM PHA-767491 to match the first incubation. (a) Cartoon of experimental set-up. (b) At the indicated times, DNA was isolated, separated on an alkali agarose gel and autoradiographed. Molecular weight markers (in kb) are shown to the left. (c) Chromatin isolated after the first incubation was immunoblotted for Mcm4 (lanes 3 and 4). Chromatin from a parallel incubation lacking aphidicolin was loaded as a comparison (lanes 1 and 2). (d–f) Sperm nuclei were incubated for 60 min in extracts treated with p27kip1 to allow Mcm4 hyperphosphorylation. PHA-767491 (50 μM) was then added, and chromatin was isolated either immediately after PHA-767491 addition (60 min) or 5 or 15 min later (65 or 75 min, respectively). Extract was optionally supplemented with the indicated concentrations of okadaic acid at the time of PHA-767491 addition (d) 1.2 μM I-2 45 min after sperm addition (e) or 1 µM tautomycetin at the time of PHA-767491 addition (f).
	Figure 3. Effects of I-2, PHA-767491 and Cdk activity on DNA replication. (a) Interphase Xenopus egg extract was supplemented with demembranated sperm nuclei, [α-32P]dATP and different concentrations of PHA-767491, plus or minus I-2; after 90 min total DNA synthesis was determined. Mean and s.e.m. of three independent experiments is shown. (b) Extract was supplemented with sperm nuclei at 15 ng DNA μl−1, [α-32P]dATP and optionally with I-2. At the indicated times, total DNA synthesis was measured. Mean and s.e.m. of three independent experiments is shown. (c) Extract was supplemented with sperm nuclei at 10 ng DNA μl−1 and optionally with I-2. At the indicated times, chromatin was isolated and immunoblotted for Mcm4 and PCNA. The lower portion of the gel was stained with Coomassie to visualize histones. (d) Chromatin was prepared by incubating sperm nuclei at 20 ng DNA μl−1 in interphase extract supplemented with 50 μM PHA-767491; after 60 min chromatin was isolated and transferred to fresh extract containing [α-32P]dATP optionally supplemented with p27kip1. At different times, aliquots were taken, and total DNA synthesis was determined. Mean and s.e.m. of three independent experiments is shown. (e) ‘KIP1 chromatin’ was prepared by incubating sperm nuclei at 20 ng DNA μl−1 in interphase extract supplemented with p27kip1 and 1.2 μM I-2; after 40 min, chromatin was isolated. ‘KIP1 chromatin’ or naive sperm nuclei were then incubated in fresh extract containing [α-32P]dATP, optionally supplemented with 20 μM PHA-767491 and 1.2 μM I-2. At different times, aliquots were taken, and total DNA synthesis was determined and expressed as percentage of the amount synthesized by 90 min in control (untreated extract). Mean and s.e.m. of three independent experiments is shown.
	Figure 4. Cdc7 fulfills its essential function early in S phase. (a,b) G1 CHO nuclei were incubated in interphase extract supplemented with geminin and optionally supplemented with I-2 or [α-32P]dATP. At the indicated times, chromatin was isolated and immunoblotted for Mcm4 (a) or DNA synthesis was determined by measuring [α-32P]dATP incorporation (b). (c–f) Interphase egg extracts were supplemented with G1 CHO nuclei, geminin and I-2 and optionally with [α-32P]dATP. At different times thereafter extract was optionally supplemented with 20 μM PHA-767491 or 1 mM roscovitine. (c) Schematic of experiments. (d,e) At the indicated times, total DNA synthesis was determined. (f) At 60 or 90 min, extract was pulsed with Cy3-dUTP and sites of DNA synthesis were visualized by fluorescence microscopy. The proportion of nuclei showing different replication patterns (I–V; representing stages from early through late S phase) was determined. The mean of three independent experiments (n = 20 nuclei for each sample) is plotted [58]. Type I, faintly punctate labelling throughout euchromatic regions; type II, complete diffuse labelling of euchromatic regions, with lack of nucleolar labelling; type III, intense labelling of the peripheral ring; type IV, labelling of small speckled heterochromatic foci within the nuclear interior or at the periphery; type V, predominant labelling of large internal replication foci and at the periphery of the nucleus.
	Figure 5. Early addition of etoposide reduces Cdc7 activity on chromatin. (a–e) Sperm nuclei were incubated at 10 ng DNA μl−1 in extracts treated with 300 μM etoposide (ETO) and/or 5 mM caffeine. (a,c) At the indicated times, chromatin was isolated and immunoblotted for Mcm4, Cdc7 and Cdc45. The lower portion of the gel was stained with Coomassie to visualize histones. (b) The percentage of Mcm4 hyperphosphorylation was quantified in three independent experiments. The mean and s.e.m. is shown. (d) Cdc7 levels were quantified and expressed as a proportion of the peak value. Mean and s.e.m. for six independent experiments are shown. (e) Extracts were also supplemented with [α-32P]dATP and after 90 min total DNA synthesis was determined. (f,g) 10 ng DNA μl−1 sperm nuclei were incubated in extracts, and at 0, 15 or 30 min after DNA addition extract was optionally supplemented with 300 μM etoposide. (f) After 90 min, nuclei were stained with Hoechst 33258 and visualized by phase contrast (top) or fluorescence microscopy (bottom). Scale bar, 25 μm. (g) At the indicated times, chromatin was isolated and immunoblotted for Mcm4, Cdc7, PCNA and Topo II.
	Figure 6. The checkpoint response to etoposide promotes Mcm4 dephosphorylation. (a–f) Sperm nuclei were incubated in extracts optionally treated with 300 μM etoposide, 5 mM caffeine, 100 nM p27kip1, 10 μM KU55933, 10 μM NU7441 and/or 1.2 μM I-2. (a,c,e) At the indicated times, chromatin was isolated and immunoblotted for Mcm4, Cdc45 or PCNA. The lower portion of the gel was stained with Coomassie to visualize histones. (b) Extract was also supplemented with [α-32P]dATP. After incubation for 90 min, the total amount of DNA synthesized was determined by scintillation counting. (d,f) The percentage of Mcm4 hyperphosphorylation was quantified in three independent experiments. The mean and s.e.m. is shown. (g,h) Sperm nuclei were incubated in extract optionally supplemented with etoposide and/or caffeine for 60 min. Extract was then supplemented with 50 μM PHA-767491, and at the indicated times thereafter chromatin was isolated and immunoblotted for Mcm4. (h) The proportion of Mcm4 that was hyperphosphorylated was quantified. Mean and s.e.m. of three independent experiments is shown. The degree of dephosphorylation induced by etoposide varied between the three experiments, but within each experiment the rate of dephosphorylation was reduced by co-addition of caffeine.
	Figure 7. Checkpoint-dependent Mcm4 dephosphorylation and PP1. (a–c) Sperm nuclei were incubated in extracts optionally treated with etoposide, aphidicolin, EcoRV and 5 mM caffeine. At the indicated times, chromatin was isolated and immunoblotted for Mcm4, PP1α, PP1γ and γH2A-X. The bottom part of each gel was stained with Coomassie to visualize histones. (d,e) Sperm nuclei were incubated in extracts optionally treated with different combinations of 0.75 µM PHA-767491, 100 µM aphidicolin and 5 mM caffeine. At the indicated times, chromatin was isolated and immunoblotted for Mcm4. The bottom part of each gel was stained with Coomassie to visualize histones. The percentage of Mcm4 hyperphosphorylation was quantified in three independent experiments; the mean and s.e.m. is shown (e).
	Figure 8. Schematic diagram of the role and regulation of Cdc7 in Xenopus egg extracts. (a) A single replication origin is shown which is licensed by loading a double hexamer of Mcm2–7 proteins (‘MCM’). This promotes ELYS binding and the recruitment of Cdc7 to Mcm2–7 at the origin. (b) Events occurring during S phase are shown at both an early-replicating (left) and a late-replicating origin (right). Mcm2–7 at both origins are phosphorylated by Cdc7, which in turn is reversed by PP1. Cdk substrates for initiation are recruited preferentially to the early-replicating origin. Checkpoint kinases activated in response to etoposide or other inhibitors promote PP1 chromatin association with reverse Cdc7 activity but may also independently inhibit Cdk activity.

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