Kundu LR , Kumata Y , Kakusho N , Watanabe S , Furukohri A , Waga S , Seki M , Masai H , Enomoto T , Tada S .

	Figure 1. DNA replication is inhibited by excess Cdc6 in a manner independent of ATM/ATR-dependent checkpoint pathways. (A) Upper panel: chromatin was isolated after incubation of sperm nuclei (3 ng DNA/µl) for 20 min with extracts containing buffer (none), geminin (100 nM) or various concentrations of GST–Cdc6. Isolated chromatin was subjected to immunoblotting. Open and closed arrowheads represent endogenous Cdc6 and GST–Cdc6, respectively. Egg extracts (Ex; 2 µl) were also applied. Lower panel: DNA synthesis was measured after a 90-min incubation of sperm nuclei (3 ng DNA/µl) with Xenopus egg extracts supplemented with various concentrations of GST–Cdc6. (B) Synthesized DNA at indicated time points was measured after incubation of sperm nuclei (3 ng DNA/µl) with extracts supplemented without (open symbol) or with GST–Cdc6 (700 nM; closed symbol). (C) Nuclei were isolated after sperm nuclei (3 ng DNA/µl) were incubated with egg extracts supplemented with aphidicolin (40 ng/µl) or GST–Cdc6 (700 nM) in the presence or absence of 5 mM caffeine. The isolated fractions were immunoblotted for phospho-Chk1 (Chk1-P) and histone H3 (H3). Egg extracts (Ex; 2 µl) were also applied. (D) DNA synthesis was measured after 90-min incubation of sperm nuclei (3 ng DNA/µl) with extracts supplemented with buffer (none), EcoRI (0.2 U/µl) or GST–Cdc6 (700 nM) in the presence or absence of 5 mM caffeine.
	Figure 2. Cdc6 inhibits DNA replication after origin licensing at an earlier stage of DNA replication. (A and B) GST–Cdc6 (700 nM) was added to the reaction mixture at indicated times after the commencement of incubation of sperm nuclei (3 ng DNA/µl) with egg extracts. Synthesized DNA was measured after 90 min from the start of incubation. (C) A schematic representation of the experimental procedures for D and E. (D) Chromatin was isolated after incubation of sperm nuclei (3 ng DNA/µl) for 20 min with extracts containing buffer (none) or GST–Cdc6 (700 nM) in the presence or absence of geminin (100 nM). Isolated chromatin was subjected to immunoblotting. Open and closed arrowheads represent endogenous Cdc6 and GST–Cdc6, respectively. Egg extracts (Ex; 2 µl) were also applied. (E) Sperm nuclei (15 ng DNA/µl) were incubated with extracts supplemented with buffer (none) or GST–Cdc6 (700 nM) for 20 min in the presence or absence of geminin (100 nM). Chromatin was isolated and incubated for 90 min with fresh geminin-supplemented extracts containing [α-32P]dATP. DNA synthesis in the second incubation was measured and represented as a percentage of the radioactivity incorporated into DNA in the samples of interest to that after incubation with control extracts for 90 min.
	Figure 3. Cdc6 halts DNA replication before Cdk2-dependent steps. (A) A schematic representation of the experimental procedures. (B) Sperm nuclei (6 ng DNA/µl) were incubated with extracts containing aphidicolin (40 ng/µl) for 60 min and isolated to obtain aphidicolin nuclei. Amount of DNA synthesized was measured during a 90-min incubation after untreated sperm chromatin (sp) or aphidicolin nuclei (ap) were added in extracts containing buffer (none), aphidicolin (aph; 40 ng/µl) or GST–Cdc6 (700 nM). DNA synthesis is represented as a percentage of the radioactivity incorporated into DNA in the samples of interest to that after incubation with control extracts for 90 min. (C) The same experiment as shown in B but using p21 (5 ng/µl) instead of aphidicolin for nuclear preparation to obtain p21 nuclei (21) and for additives of the second incubation (p21).
	Figure 4. Cdc6 inhibits DNA replication after Cdc7 has been loaded. (A) Sperm nuclei (3 ng DNA/µl) were incubated in extracts supplemented with buffer (none) or GST–Cdc6 (700 nM). Chromatin was isolated at indicated time points and subjected to immunoblotting. Egg extracts (Ex; 2 µl) were also applied. (B) DNA synthesis in sperm nuclei were measured after incubation for 90 min in non-treated extracts (column 1), extracts supplemented with 700 nM of GST–Cdc6 (column 2) or Cdc7-depleted (ΔCdc7) extracts (column 3). Sperm nuclei (15 ng DNA/µl) were incubated in extracts supplemented without (non-treated) or with 700 nM of GST–Cdc6 (+GST–Cdc6) for 45 min, followed by nuclear isolation. DNA synthesis was assayed after the nuclei isolated from non-treated or GST–Cdc6 supplemented extracts were incubated for 90 min with Cdc7-depleted extracts (ΔCdc7) containing 100 nM of geminin (column 4 or 5, respectively). DNA synthesis is represented as a percentage of the radioactivity incorporated into DNA in the samples of interest to that after incubation with control extracts for 90 min. Inset: Mock-treated (m) and Cdc7-depleted (Δ) extracts (Ex; 2 µl) were subjected to immunoblotting. (C) Sperm nuclei (15 ng DNA/µl) were incubated for 45 min in extracts supplemented with buffer (none) or GST–Cdc6 (700 nM) and were isolated (lanes 1 and 2). The isolated nuclei were incubated with Cdc7-depleted extracts supplemented with geminin (100 nM), and the chromatin-bound proteins at indicated time points were detected by immunoblotting (lanes 3–6). Open and closed arrowheads represent endogenous Cdc6 and GST–Cdc6, respectively.
	Figure 5. Cdc6 inhibits Cdc7-dependent phosphorylation of Mcm4. (A) Chromatin was isolated at 30 and 45 min from extracts supplemented with buffer (none) or GST–Cdc6 (Cdc6; 700 nM) and subjected to λ-PPase treatment in the presence of absence of EDTA, followed by immunoblotting analysis. Egg extracts (Ex; 2 µl) were also applied. (B) Chromatin was isolated after a 45-min incubation of sperm nuclei (3 ng DNA/µl) with extracts supplemented with buffer (none), GST–Cdc6 (Cdc6; 700 nM), p21 (5 ng/µl), caffeine (caff; 5 mM) or wortmannin (wort; 100 µM) and subjected to immunoblotting. Egg extracts (Ex; 2 µl) were also applied. (C) Chromatin was isolated after a 75-min incubation of sperm nuclei (15 ng DNA/µl) with mock-treated (mock) or Cdc7-depleted (ΔCdc7) extracts. Isolated chromatin was treated with λ-PPase in the presence or absence of EDTA, and then subjected to immunoblotting. Mock-treated (m) and Cdc7-depleted (Δ) extracts (Ex; 2 µl) were also applied.
	Figure 6. Cdc6 inhibits Cdc7-dependent phosphorylation of Mcm2–7 on chromatin during DNA replication. (A) Silver staining (upper) and autoradiography (lower) profiles of the kinase assay using recombinant proteins. Mouse Mcm2–4–6–7 complex was incubated at 30°C for 60 min without (lanes 7 and 8) or with human Cdc7–ASK (lanes 1–6) in the absence (lanes 7 and 8) or presence of GST–Cdc6 (160, 320, 480, 640, 800 and 800 nM for lanes 2, 3, 4, 5, 6 and 7, respectively). (B) Chromatin was isolated after 0-, 15-, 30- or 45-min incubation of sperm nuclei (3 ng DNA/µl) with extracts supplemented with buffer (none) or GST–Cdc6 (700 nM). Isolated chromatin was subjected to immunoblotting.
	Figure 7. Outline of the observations leading to the inhibition of DNA replication by deregulated Cdc6. (A) Cdc6 is targeted to origin after chromatin binding of ORC. (B) Mcm2–7 is recruited to chromatin by Cdt1. (C) After Mcm2–7 has been loaded onto chromatin (licensing), Cdc6 is destabilized from chromatin. (D) Origin firing occurs by Cdc7 (DDK)-dependent phosphorylation of Mcm2–7 in concert with Cdk2 kinase activity. (E) When Cdc6 is deregulated resulting in insufficient removal from chromatin after licensing, DNA replication is arrested after chromatin loading of DDK. (F) Persisting Cdc6 on chromatin after origin licensing prevents Cdc7-dependent phosphorylation of Mcm2–7 thereby blocking further steps.

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