Hua XH , Newport J .

GM 44656 NIGMS NIH HHS

	Figure 2. After being displaced from chromatin by cyclin A, ORC does not reassociate with chromatin after forming nuclei. The reaction was carried out as in Fig. 1 A except membrane was added at the end of the reaction. The samples were incubated for a further 60 min to assay chromatin-bound MCM3, ORC2, and DNA replication. (A) Nuclei were pelleted through a sucrose cushion, permeabilized, and then spun again through a second cushion (see Materials and Methods). Pellet fractions were recovered and chromatin-bound MCM and ORC determined by Western analysis. After forming nuclei, ORC remains displaced from chromatin. The small amount of ORC observed with early and late addition are likely to be the ORC nonspecifically associated with membrane. To prevent MCM dissociation from chromatin as a result of replication, aphidicolin (50 μg/ml) was added in these reactions. (B) DNA replication assay. The incorporation of radioactive-labeled dATP is identical between control and late cyclin A–treated extract.
	Figure 3. MCM-associated, ORC(−) chromatin can replicate in an ORC-depleted extract. (A) Diagram of the experiment in (C). Early and late addition of cyclin A produces two types of sperm. When cyclin A is added before sperm (early addition), neither MCM (M) nor ORC (O) can bind to chromatin. This treatment results in sperm lacking MCM and ORC (top). When cyclin A is added after sperm (late addition), only ORC dissociates from chromatin. This treatment produces sperm containing MCM but lacking ORC (bottom). These two types of sperm were isolated by centrifugation though a sucrose cushion. After removing the supernatant and sucrose cushion, sperm pellets were resuspended in either mock-depleted (1) or ORC-depleted extract (2 and 3), and DNA replication was assayed. (B) Western blot showing the depletion of ORC2 from the extract. Anti-ORC antibodies were coupled to protein A–Sepharose beads and incubated with interphase extract for 45 min. The beads were removed by low speed centrifugation. After three cycles of depletion, >96% of the ORC was removed. Preimmune serum was used as control (mock depl.). (C) Replication assays described in A were carried out for 90 min, and incorporation of radioactive dATP was quantitated. MCM (+) chromatin replicates efficiently in ORC-depleted extract (3). MCM (−) chromatin replicates well only in mock-depleted (1) but not in ORC-depleted extract (2).
	Figure 4. Chromatin-bound cdc6 is rapidly degraded and does not reassociate with DNA until after initiation of replication. (A) 2,000/μl of sperm chromatin was added into interphase extract and incubated for 5, 10, and 20 min. Sperm was then pelleted and associated cdc6 was analyzed by Western blot (chromatin-bound cdc6; 5′, 10′, and 20′). Cdc6 associates with chromatin at 5 min, and completely disappears from chromatin after 20 min. Alternatively, interphase cytosol was preincubated with HisUbR48 (R48), His-Ub (WT-Ub), or His-Cip (Cip) for 20 min, and then sperm chromatin was added and incubated for a further 30 min. Chromatin-associated cdc6 is stabilized by UbR48 and Cip but not by WT-Ub. His-cdc6 is shown as a standard (ST.). (B) Interphase extract was incubated with sperm chromatin and membrane with or without aphidicolin. After the indicated period of time, the reactions were stopped by diluting fivefold with ELB and pelleting through a sucrose cushion. The nuclei were permeabilized and pelleted again. Chromatin-associated cdc6 and MCM were analyzed by Western blotting using specific antibodies. (C) Sperm and membrane were added into interphase cytosol and DNA replication was allowed to occur for the indicated time periods (t = 0 represents aphidicolin arrest). For each time point, the sample was divided into two parts, and different extraction methods were used to determine chromatin-associated MCM3 and ORC2 (see Materials and Methods). Dissociation of MCM3 from chromatin during DNA replication can be seen only with the high stringency wash. (D) Interphase cytosol was incubated alone (−R48) or with R48 (+R48) for 20 min. Sperm, membrane, and aphidicolin (50 μg/ml) were then added to both and the reactions incubated for a further 60 min. After this, both reactions were split into two parts. Nuclei were extracted using high stringency condition for one part and low stringency condition for the other. Chromatin-associated cdc6 was then assayed by Western blot. With a high stringency wash, cdc6 cannot be detected on chromatin in absence of R48. In the low stringency wash of the same nuclei, significant amounts of cdc6 can be seen attached to chromatin both with and without R48.
	Figure 5. Inhibition of protein degradation prevents DNA replication. (A) Interphase cytosol was incubated with either R48 (+R48 early) or with sperm chromatin (+R48 late) for 20 min. Then sperm chromatin or R48 was added, respectively, and the samples were incubated for another 20 min. After this, membrane was added to both, and DNA replication at indicated time points was assayed. DNA replication with early addition of R48 is strongly inhibited, whereas replication with late addition is identical to the control (no addition). (B) The effect of R48 can be reversed by dilution. 20 μl of interphase cytosol was incubated with R48 for 20 min. Sperm was then added and incubated for another 30 min. After this the reaction was split into two halves and 90 μl of fresh cytosol was added into only one (Dil. R48). The samples were then assayed for both cdc6 content and DNA replication. 10-fold dilution of R48 destabilizes chromatin-bound cdc6 and restores DNA replication.
	Figure 6. After MCM binds to chromatin, DNA replication is still inhibited by Cip. (A) Western blot showing the effect of Cip on MCM3 and ORC2 binding. Interphase cytosol was incubated either with buffer (−Cip) or with Cip (+Cip) for 20 min. Sperm (5,000/μl) was added and incubated for a further 30 min. The samples were then diluted and pelleted through a sucrose cushion. Pellet fractions were recovered and subject to Western blotting analysis using specific antibodies. The result shows that the binding of ORC and MCM to chromatin is insensitive to Cip addition. (B) An illustration of the DNA replication assay performed in C. Interphase cytosol was preincubated with sperm (5,000/μl) for 30 minutes (step 1). 100 nM of cyclin A was then added and incubated a further 30 min (step 2). This treatment removes prebound ORC (O) without displacing MCM (M). Sperm chromatin was then isolated from the cytosolic mixture by centrifugation through a sucrose cushion (step 3). This sperm chromatin was then resuspended in ORC-depleted extract (step 4). The sample was then divided into two parts and Cip was added to only one of them. DNA replication was then assayed. (C) DNA replication assay showing incorporation of radioactive dATP at 30, 60, and 90 min after adding ORC-depleted extract. Under these experimental conditions DNA replication is greatly inhibited by Cip.
	Figure 7. Steps leading to initiation of replication. During early G1, cdk2 kinase activity is absent and ORC, cdc6, and MCM proteins bind to chromatin in a sequentially dependent manner to generate preinitiation complexes. During late G1, high levels of cdk2 activity accumulate. This activity leads to the degradation of cdc6. We have shown that activation of moderate levels of cdc2– cyclin A kinase at this time leads to displacement of ORC, creating an initiation complex containing only MCM. After cdc6 is displaced from preinitiation sites there is a second cdk2-dependent step that must occur before actual initiation takes place.

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