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FIGURE 1. Protein release from replicating X. laevis chromatin. A, schematic representation of chromatin protein preparation procedure. B and C, chromatin was isolated from egg extract in the middle of S-phase (when replisome proteins peak on chromatin) in the presence of 50 mm potassium acetate, and DNA was digested by Benzonase and sonication. Ex, 0.5 μl of egg extract; Ch, chromatin after first centrifugation; other lanes correspond to material isolated from 5 μl of egg extract. B, chromatin was maintained in 50 mm potassium acetate throughout the procedure. P, pellet after DNA digestion; S, supernatant after DNA digestion. C, after DNA digestion, the salt concentration was adjusted to the 50, 100, 150, 200, 250, 300, 350, or 400 mm potassium acetate before samples were separated by centrifugation into soluble and insoluble (pellet) fractions. Fractions were separated by SDS-PAGE and immunoblotted with antibodies to the indicated proteins.
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FIGURE 2. Mcm2-7 form large complexes on chromatin before replication initiation. Chromatin was isolated from egg extract plus or minus 100 nm p27KIP1 and chromatin proteins released in 50 mm potassium acetate. A, Ex, 0.5 μl egg extract; Ch, chromatin after first centrifugation; other lanes correspond to insoluble pellet (P), and soluble chromatin (S) from 5 μl extract. B and C, top panels: chromatin was isolated from extracts treated with p27KIP1 and soluble proteins released in 50 mm potassium acetate were subjected to gel filtration (B) or glycerol gradient fractionation (C) and immunoblotted with antibodies to Mcm2-7. Bottom panels: extract incubated with 100 nm p27KIP1 was subjected to gel filtration (B) or glycerol gradient fractionation (C) in buffer containing 50 mm potassium acetate and immunoblotted with antibodies to Mcm2-7. D, data from B and C were used to determine the molecular weight of Mcm2-7 complexes in whole extract and in chromatin. E, chromatin in mid S phase was isolated from extracts without added p27KIP1 and soluble proteins separated by gel filtration as for B.
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FIGURE 3. Mcm2-7 complexes immunoprecipitated from chromatin assembled in egg extract with blocked CDK activity look the same as Mcm2-7 at unfired origins in S-phase. Chromatin was isolated from egg extract plus or minus 100 nm p27KIP1 and chromatin proteins were released in 50 mm potassium acetate. Soluble protein was then separated by gel filtration. A and B, high molecular weight fractions containing Mcm2-7 were pooled and immunoprecipitated with either non-immune or anti-Mcm3 antibodies. Immunoprecipitates were separated by SDS-PAGE and gels were stained with colloidal Coomassie stain. C, gel shown in B was sliced into 40 segments, and each segment was subjected to mass spectrometry. The spectral count for each identified protein was summed over all 40 gel slices. The top 20 identified proteins specific for the Mcm3 immunoprecipitate are shown, along with its spectral count. D, each of the gel filtration fractions were analyzed by mass spectrometry, and the abundance of proteins estimated by total ion current. For the 20 proteins shown in C, the relative protein abundance across the gel filtration column is shown as a heat map. Red, peak abundance; black, half-peak abundance; green, no protein detected.
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FIGURE 4. Large Mcm2-7 complexes form independently of Cdc7 kinase activity and are substrates of Cdc7 kinase. A, chromatin was isolated from egg extract containing 100 nm p27KIP1 and chromatin proteins released in 50 mm potassium acetate. Soluble protein was then separated by gel filtration. In parallel, whole extract was separated by gel filtration under identical conditions. Aliquots of chromatin C and extract E fractions were separated by SDS-PAGE and immunoblotted for Mcm2 and Mcm4. B, soluble chromatin proteins were isolated in 50 mm potassium acetate from extract optionally supplemented with 100 nm p27KIP1 or 100 μm PHA-767491, separated by SDS-PAGE and immunoblotted for the indicated proteins. An aliquot of untreated extract is also shown. C, chromatin from extract treated with p27KIP1 or PHA-767491 was prepared as in B and separated by gel filtration. Fractions were separated by SDS-PAGE and immunoblotted for the indicated proteins.
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FIGURE 5. Mid-S phase chromatin. A, chromatin was isolated from egg extract in the middle of S-phase (when replisome proteins peak on chromatin), and proteins were optionally released from chromatin with benzonase in the presence of 50 or 300 mm potassium acetate as indicated. Ex, 0.5 μl of egg extract; Ch, chromatin after first centrifugation; other lanes correspond to insoluble pellet (P) and soluble chromatin (S) from a 5-μl extract after a second centrifugation. B and C, protein complexes released from mid-S phase chromatin by benzonase and solubilized in 300 mm potassium acetate were separated by gel filtration (B) or glycerol gradient (C). Fractions were separated by SDS-PAGE and immunoblotted with antibodies to the indicated proteins.
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FIGURE 6. Xenopus CMG complex. Chromatin was isolated from egg extract in the middle of S-phase (when replisome proteins peak on chromatin), and proteins were released from chromatin in presence of 50, 100, or 300 mm potassium acetate. A, soluble chromatin protein complexes were separated by gel filtration and glycerol gradient and CMG components (Mcm2-7, Cdc45, and GINS) were followed through fractionation by immunoblotting. The data were used (19) to determine the molecular weight of the complexes. B and C, mid-S phase chromatin solubilized in 300 mm potassium acetate was immunoprecipitated with non-immune or anti-Cdc45 antibodies. Proteins present in the immunoprecipitates were analyzed by immunoblotting (B) or by mass spectrometry (C).
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FIGURE 7. Behavior of other replisome proteins. Chromatin was isolated from egg extract in the middle of S-phase (when replisome proteins peak on chromatin) and proteins were released from chromatin in 100 mm potassium acetate. A, soluble chromatin protein complexes were separated by gel filtration and immunoblotted for a range of replisome proteins. B, mid-S phase chromatin solubilized in 100 mm potassium acetate was immunoprecipitated with non-immune, anti-Cdc45 or anti-Ctf4 antibodies. Proteins present in the immunoprecipitates were analyzed by immunoblotting. C, mid-S phase chromatin solubilized in 100 mm potassium acetate was immunoprecipitated with non-immune and anti-Cdc45 antibodies, and the precipitates were analyzed by mass spectrometry.
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FIGURE 8. CMG complex at minimally licensed chromatin. Mcm3 depleted extract was supplemented with 10% of normal extract. A, DNA synthesis was assayed by α-[32P]dATP incorporation. B, chromatin was isolated at the indicated times and immunoblotted for Mcm2 and Cdc45 proteins. C, chromatin was isolated at 45 min and proteins were released from chromatin in 100 mm potassium acetate. Soluble chromatin protein complexes were separated by gel filtration and immunoblotted for the indicated proteins.
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