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Figure 1. Stable association of Cdc6 with chromatin is abolished after licensing. (A) Sperm nuclei were incubated at 10 ng DNA/μl in interphase extract. At the indicated times chromatin was isolated and immunoblotted for Orc2, Cdc6, Mcm5, Cdt1, and geminin. As controls, extract was incubated for 30 min without sperm, or was supplemented with gemininDEL before incubation for 30 min with sperm. (B) Coomassie-stained gel of recombinant Cdt1 and purified Mcm2-7. (C and D) Unlicensed chromatin was isolated from extract supplemented with recombinant gemininDEL, and was incubated for 30 min with recombinant Cdt1 and/or purified Mcm2-7 or in untreated extract. Chromatin was (C) transferred to extract supplemented with recombinant gemininDEL and α-[32P]dATP, and subsequent DNA synthesis was assayed; or (D) isolated and immunoblotted for Orc2, Cdc6, Cdt1, Mcm2, Mcm4, Mcm5, Mcm6, and histone H3.
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Figure 2. Unlicensed chromatin becomes saturated with approximately two copies of Cdc6 every 10 kb. 400 ng of aliquots of sperm nuclei were incubated for 30 min in increasing volumes of interphase extract supplemented with gemininDEL. (A) Chromatin was isolated and immunoblotted for Cdc6. Lanes 1–8 represent chromatin incubated in 80, 60, 40, 20, 10, 4, 2, and 1 μl extract. Lane 9 shows a mock chromatin isolation from 20 μl extract containing no DNA. Lanes 10–14 show loading standards, containing 5, 10, 15, 20, and 25 ng recombinant Cdc6. (B) The quantity of chromatin-bound Cdc6 was estimated from gels as shown in A. The mean and SD of three separate experiments is shown.
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Figure 3. Cdc6 is destabilized on chromatin after the minimal effective quantity of Mcm2-7 is loaded. (A and B) Sperm nuclei were incubated at 10 ng DNA/μl in interphase egg extract, and gemininDEL was added at the indicated times after sperm addition. (A) Chromatin was isolated 5 min after gemininDEL addition and immunoblotted for Cdc6 and Mcm2. (B) DNA synthesis was assayed by α-[32P]dATP incorporation. (C) Sperm nuclei were incubated in interphase egg extract for 4 min; chromatin was isolated and incubated for an additional 0 or 26 min in extract previously immunodepleted of ORC or Cdc6 or mock depleted with nonimmune antibodies (NI−). Chromatin was isolated and immunoblotted for Mcm2. Chromatin isolated after a 30-min incubation in untreated extract is shown as control. The top shows a schematic diagram of the experimental protocol.
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Figure 4. Cdc6 reloading onto chromatin starts at the beginning of S phase. Sperm nuclei were incubated at 7 ng DNA/μl in interphase egg extract. (A) Time course of DNA synthesis. (B and C) Chromatin was isolated at the indicated times and immunoblotted for PCNA, Cdc6, Mcm2, Cdt1, and geminin. The immunoblots are shown in B and quantitation of the intensity of Mcm2, PCNA, and Cdc6 is shown in C. (D) Chromatin from either untreated extract or extract supplemented with geminin was isolated at the indicated times and immunoblotted for Mcm2 and Cdc6.
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Figure 5. Cdc6 reloading onto chromatin is dependent on the initiation of replication. Sperm nuclei were incubated at 10 ng DNA/μl in interphase egg extract supplemented with combinations of 1 mM of roscovitine, 5 mM of caffeine, DMSO, or different concentrations of aphidicolin. After 90 min, samples were assayed for DNA synthesis (A and C) or chromatin was isolated and immunoblotted for Cdc6 and Mcm2 or Mcm7 (B and D).
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Figure 6. Cdc6 reloading requires fork elongation. Sperm nuclei were incubated at 10 ng DNA/μl in interphase egg extract treated with 0–80 μM aphidicolin in the presence of 5 mM of caffeine and α-[32P]dATP. (A) At 90 min, chromatin was isolated and immunoblotted for Mcm2, PCNA, and Cdc6. (B) DNA was separated on an alkaline agarose gel and autoradiographed. The migration of molecular weight markers (in bp) is shown on the left.
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Figure 7. Cdc6 reloading is ORC dependent and Cdc6 is required in S phase for Chk1 activation. (A) Schematic of the experimental procedure. Sperm nuclei were incubated for 15 min at 15 ng DNA/μl in interphase egg extract. Chromatin was isolated under high salt conditions to remove ORC. Chromatin was incubated in a second extract depleted of either ORC or Cdc6 or mock depleted with nonimmune antibodies (NI−). DNA synthesis in the second extract (without aphidicolin or caffeine) was measured by incorporation of α-[32P]dATP (B, D, and F). The second extract was optionally supplemented with combinations of 40 μM aphidicolin or 5 mM of caffeine, and after 90 min in the second extract, chromatin, or intact nuclei were isolated and immunoblotted with the indicated antibodies (C, E, and G). (B and C) Licensed high salt-washed chromatin was incubated in Orc1- or nonimmune-depleted extracts. Chromatin was isolated and immunoblotted for Orc1, Cdc6, and Mcm2. (D and E) Licensed high salt-washed chromatin was incubated in Cdc6- or nonimmune-depleted extracts. Nuclei were isolated and immunoblotted for Orc1, Cdc6, Chk1, and phospho-Chk1. (F and G) Licensed high salt-washed chromatin was incubated in Orc1- or nonimmune-depleted extracts. Nuclei were isolated and immunoblotted for Orc1, Cdc6, Chk1, and phospho-Chk1.
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Figure 8. The checkpoint function of Cdc6 does not depend on Mcm2-7 loading. (A) Sperm nuclei were incubated in extract supplemented with 40 μM aphidicolin. At 15 min, after licensing had occurred, recombinant gemininDEL was added to an aliquot of the reaction. At 30, 60, 90, 120, and 150 min from the start of the incubation, nuclei were isolated and immunoblotted for Chk1 and phospho-Chk1. Nuclei isolated at 60 min from extract lacking aphidicolin is also shown as control. (B) Sperm nuclei were incubated for 15 min at 15 ng DNA/μl in interphase egg extract. Chromatin was isolated under high salt conditions to remove ORC. Chromatin was then incubated in a second extract depleted of Cdc6 or mock depleted with nonimmune antibodies (NI−). Both extracts were supplemented with 40 μM aphidicolin. At 0, 1, 2, 3, or 4 h afterwards, chromatin was isolated and immunoblotted for Mcm2, Cdc45, RPA-70, or PCNA.
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Figure 9. A model for Cdc6 function. (A) Dimers of Cdc6 bind tightly to unlicensed chromatin. (B) Although 10–20 copies of Mcm2-7 are normally loaded onto each origin, loading of the first one to two copies of Mcm2-7 (M) is sufficient to minimally license the origin. Once this has happened, the affinity of Cdc6 for the origin declines. This potentially directs Cdc6 and Mcm2-7 to other unlicensed origins. (C) Once replication forks have initiated and moved a small distance from the origin, Cdc6 can rebind to ORC at the origin with high affinity. This permits recruitment of cyclin E to the origin. Cdc6 is also required during S phase to allow Chk1 activation in response to replication fork inhibition, though ORC-dependent chromatin binding is not required for this function.
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