PMC:548366 / 5545-15595 JSONTXT

{"project":"2_test","denotations":[{"id":"15687385-9251047-77096023","span":{"begin":177,"end":179},"obj":"9251047"},{"id":"15687385-9251047-77096024","span":{"begin":394,"end":396},"obj":"9251047"},{"id":"15687385-10766247-77096025","span":{"begin":646,"end":647},"obj":"10766247"},{"id":"15687385-9635433-77096026","span":{"begin":1021,"end":1023},"obj":"9635433"},{"id":"15687385-9251047-77096027","span":{"begin":1648,"end":1650},"obj":"9251047"},{"id":"15687385-7760937-77096028","span":{"begin":2001,"end":2003},"obj":"7760937"},{"id":"15687385-9251047-77096029","span":{"begin":2004,"end":2006},"obj":"9251047"},{"id":"15687385-8861912-77096030","span":{"begin":2007,"end":2009},"obj":"8861912"},{"id":"15687385-9251047-77096031","span":{"begin":6845,"end":6847},"obj":"9251047"},{"id":"15687385-12118071-77096032","span":{"begin":6848,"end":6850},"obj":"12118071"},{"id":"15687385-9251047-77096033","span":{"begin":7876,"end":7878},"obj":"9251047"},{"id":"15687385-12118071-77096034","span":{"begin":7879,"end":7881},"obj":"12118071"}],"text":"MATERIALS AND METHODS\n\nPreparation of Xenopus egg extract\nExtracts derived from metaphase-arrested Xenopus eggs were prepared according to the method described by Chong et al. (31). The extract was used after a release to interphase by an addition of 0.3 mM CaCl2 and 15 min incubation at 23°C. Xenopus sperm nuclei were prepared after demembranation with lysolecithin as described previously (31).\n\nProduction of recombinant proteins\nRabbit polyclonal Cdt1 antibody was raised against recombinant full-length Xenopus Cdt1 fused with a His6-tag (His-Cdt1). A plasmid expressing His-Cdt1 was generously provided by Drs D. Maiorano and M. Mechali (5). Rabbit polyclonal Cdc6 antibody was raised against His6-tagged full-length Xenopus Cdc6 expressed in BL21-Codon Plus RIL (Stratagene). His6-tagged-geminin was expressed from Xenopus geminin H cDNA lacking a destruction box and purified using Ni-NTA agarose (Qiagen). A plasmid expressing the recombinant protein was generously provided by Drs T. McGarry and M. Kirshner (22). Xenopus Cdt1 fused with glutathione-S-transferase (GST-Cdt1) was expressed in BL21-Codon Plus RIL and purified using Glutathione-Sepharose (Amersham Biosciences). GST-Cdt1, in contrast to His-Cdt1, was efficiently recovered in a soluble fraction of E.coli lysate, and thus was used as an active protein. The RLF-B activity of GST-Cdt1 was verified by measuring the ability to supplement licensing activity in a Cdt1-depleted Xenopus egg extract.\n\nPurification from Xenopus egg extract\nThe egg extract for purification was prepared after in vivo activation of Xenopus eggs by an addition of A23187 as described previously (31). Fifty percent (w/v) polyethylene glycol 6000 (PEG) (Wako) in 20 mM HEPES-KOH, pH 8 was added to the extract to make 5% PEG to obtain precipitated and supernatant fractions. A partially purified fraction containing Cdc6 and Cdt1 (BPAS fraction) was obtained from the 5% PEG precipitated fraction by a process based on a method described previously (16,31,32). In brief, the precipitated fraction was solubilized in T'LFB1 (40 mM HEPES-KOH, pH 8.0, 20 mM potassium phosphate, 2 mM MgCl2, 1 mM EGTA, 2 mM DTT, 10% (w/v) sucrose, 0.01% (v/v) Triton X-100, 1 μg/ml pepstatin and 1 μg/ml leupeptine) supplemented with 150 mM KCl, and adsorbed onto phosphocellulose resin (P11, Whatman). The adsorbed proteins were eluted with T'LFB1 containing 500 mM KCl. The BPAS fraction was obtained by 40% saturated ammonium sulfate precipitation from the phosphocellulose eluate and was solubilized in T'LFB1. A partially purified Cdc6 fraction was prepared from the BPAS fraction after removal of Cdt1 as indicated in ‘Immunodepletion’.\nMcm2-7 complex was partially purified from the 5% PEG supernatant fraction. The fraction was subjected to further precipitation by 12% PEG, and the 5–12% PEG precipitated fraction was adsorbed by Q-Sepharose FF (Amersham Biosciences) in T'LFB1 containing 100 mM KCl. The adsorbed proteins were eluted with T'LFB1 containing 350 mM KCl, and precipitated with the addition of an equal volume of 50% PEG. The purified fraction was solubilized with T'LFB1 and stored at −80°C prior to use.\n\nIsolation of chromatin fraction\nFor immunoblotting experiments, demembranated sperm nuclei were incubated with Xenopus egg extract at 23°C for a given period. The extract was diluted in 1 ml of nuclear isolation buffer (NIB; 50 mM HEPES-KOH pH 7.6, 50 mM KCl, 2 mM DTT, 2 mM MgCl2, 0.5 mM spermidine, 0.15 mM spermine, 1 μg/ml pepstatin and 1 μg/ml leupeptin) supplemented with 0.1% Triton X-100 and 2.5 mM ATP (TNIBA), and 15% (w/v) sucrose in TNIBA (100 μl) was layered under the diluted extract. The chromatin fraction was precipitated at 9000 g in a swing bucket centrifuge for 5 min at 4°C. After a wash with 1 ml of TNIBA, the chromatin precipitate was subjected to SDS-10% polyacrylamide gel electrophoresis for immunoblotting.\nFor biochemical assays to detect licensing activity, chromatin after an incubation with the Xenopus egg extract and/or samples were diluted in 1 ml of NIB supplemented with 0.01% Triton X-100 and 2.5 mM ATP, and 100 μl of the same buffer containing 15% sucrose was layered under the diluted extract. The chromatin was then precipitated at 6000 g in a swing bucket centrifuge for 5 min at 4°C. The upper layer and supernatant of the lower layer were carefully removed until 20 μl of the buffer remained in the bottom of the tube. After resuspension by gentle pipetting, 2 μl of the isolated chromatin fraction was used for the licensing assay.\n\nImmunodepletion\nImmunodepletion was performed using anti-Cdt1 or anti-Cdc6 antibody incubated with protein A-Sepharose Fast Flow (Amersham Pharmacia), and washed with 100 mM HEPES-KOH, pH 8.0 for 1 h at room temperature. After the wash, the resultant Sepharose resin was incubated with 0.25 mM phenylmethylsulfonylfluoride for 15 min at room temperature, and thoroughly washed with LFB2 (40 mM HEPES-KOH, pH 8.0, 20 mM potassium phosphate, 2 mM MgCl2, 1 mM EGTA, 2 mM DTT, 10% (w/v) sucrose, 2.5 mM ATP, 1 μg/ml pepstatin and 1 μg/ml leupeptine) supplemented with 50 mM KCl to obtain an antibody-coupled Sepharose. The Xenopus egg extract was incubated with the same volume of the antibody-coupled Sepharose for 1 h at 4°C. The extract after removal of the Sepharose resin was used as a depleted extract.\nThis protocol was repeated a second time for Cdc6-depletion. Although it was not necessary to repeat the process for Cdt1-depletion, the protocol was performed twice to prepare Cdt1-depleted extract when it was to be compared with Cdc6-depleted extract. Where the number of rounds of Cdt1-depletion needs to be clarified, we refer to the extract obtained after a single round of immunodepletion as ‘Cdt1-single depleted extract’ and that obtained after two rounds as ‘Cdt1-double depleted extract’.\nThe removal of antigen proteins from protein fractions was also performed using the same depletion protocol. A Cdt1-free Cdc6 fraction was prepared by Cdt1-depletion from the BPAS fraction instead of the egg extract. It was verified that the fraction was able to complement the licensing activity in the Cdc6-depleted extract, but not in the Cdt1-depleted extract.\n\nMeasurement of DNA replication in Cdt1-depleted extract\nDemembranated Xenopus sperm nuclei (100 000 nuclei) were incubated at 23°C for 20 min with 20 μl of depleted extract. After the incubation, the chromatin fraction was isolated from each extract and 2 μl of it was incubated at 23°C for 3 h with 10 μl of mock-treated or Cdt1-single depleted extract supplemented with 250 μg/ml cycloheximide, 25 mM phosphocreatine, 15 μg/ml creatine phosphokinase and [α-32P]dATP (20 kBq). Total DNA synthesis during the second incubation was measured as the radioactivity incorporated into a fraction insoluble in 5% TCA as described previously (31,33). DNA synthesis is represented as a percentage of the radioactivity incorporated into DNA in the Cdt1-depleted extract to that in the mock-treated extract.\n\nLicensing assay\nTo assay RLF-B activity in Cdt1-fractions, sperm nuclei (10 000 nuclei) were incubated for 20 min at 23°C with 2 μl of Cdt1-single depleted extract after a 2-fold dilution with LFB2 supplemented with 0.01% Triton X-100 (T'LFB2) containing the Cdt1-fractions to be tested. After the incubation, 10 μl of a fresh egg extract supplemented with 250 μg/ml cycloheximide, 25 mM phosphocreatine, 15 μg/ml creatine phosphokinase, 0.5 μM recombinant geminin and [α-32P]dATP (20 kBq) was added to the reaction mixture. Geminin prevented further licensing, and therefore DNA replication during the following incubation period depended on the licensing activity in the first reaction mixture. Total DNA synthesized over a further 90 min incubation at 23°C was measured as the radioactivity incorporated into a fraction insoluble in 5% TCA as described previously (31,33).\nFor the assays indicated in Figures 2B and 5B, 15 μl of the chromatin fraction after the ‘1st incubation’ was isolated and incubated for 20 min at 23°C with 30 μl of depleted extract or protein fraction for the ‘2nd incubation’. Then, 2 μl of the reaction mixture after the 1st or 2nd incubation was mixed with 10 μl of fresh egg extract supplemented with 250 μg/ml cycloheximide, 25 mM phosphocreatine, 15 μg/ml creatine phosphokinase, 0.5 μM recombinant geminin and [α-32P]dATP (20 kBq) in the presence or absence of recombinant geminin (0.5 μM) without further isolation of the chromatin. DNA synthesis in the absence of geminin was taken as an index of the recovery of chromatin during the isolation process, and thus, licensing activity was given as a percentage of DNA synthesis in the geminin-containing extract to that in the geminin-free extract.\n\nElution of Cdt1 from chromatin\nThe chromatin fraction was isolated after the incubation of sperm nuclei (1 000 000 nuclei) with 200 μl of Xenopus egg extract for 20 min or 60 min at 23°C. Proteins bound on the chromatin were eluted by incubation of the isolated chromatin for 10 min on ice with 10 μl of T'LFB2 containing 300 mM KCl and 0.1% BSA. The eluted fraction was obtained as a supernatant after centrifugation at 9000 g for 10 min at 4°C, and stored at −80°C. The amount of Cdt1 in the eluted fraction was verified by immunoblotting analysis using known amounts of His-Cdt1 as authentic samples.\n\nImmunoblotting\nSamples for immunoblotting were electrophoresed on an SDS–10% polyacrylamide gel and electrically transferred onto a Hybond-P PVDF-membrane (Amersham Biosciences). The membrane was blocked with 10% skim milk in phosphate-buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl, 8.04 mM Na2HPO4 and 1.47 mM KH2PO4) and incubated with a primary antibody diluted 1000- to 3000-fold with PBS containing 0.05% Tween-20 and 3% BSA. After a wash with 0.05% Tween-20–PBS three times and incubation with horse-radish peroxidase-conjugated anti-rabbit or anti-mouse IgG, protein bands that reacted with the primary antibody were visualized with ECL western blotting detection reagents (Amersham Biosciences)."}