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reaction mixtures (25 μl) contained 25 mM HEPES–NaOH (pH 7.5), 50 mM sodium acetate, 10 mM Mg(CH3COO)2, 1 mM DTT, 0.25 mg/ml BSA, 0.5 mM ATP-γ-S, 0.5 mM CaCl2, 10 fmol of 32P-labeled bubble substrate, and the indicated amount of Mcm4/6/7 proteins. After incubation at 30°C for 30 min, the indicated amount of DNase I (TAKARA Biomedical, Japan) was added, and the mixtures were incubated at room temperature for 1 min. DNase I was inactivated by the addition of 250 μl of stop solution (20 mM EDTA, 0.2M sodium chloride, 1% SDS and 12.5 μg/ml yeast RNA). For nuclease P1 footprinting assay, binding reactions were carried out under the same conditions except that CaCl2 was omitted. P1 nuclease (0.3 U, Roche) was added and incubation was continued at 37°C for 1 min. Cleavage was stopped by addition of 50 μl of 25 mM EDTA and 0.4 M NaOH; the mixture was incubated at room temperature for 10 min followed by the addition of 12 μl of 3 M sodium acetate (pH 4.8). Proteins were removed by phenol–chloroform extraction, and DNAs were collected by ethanol precipitation followed by wash with 70% ethanol. The digested DNA was subjected to electrophoresis through a 10 or 12% polyacrylamide sequencing gel containing 7 M urea, followed by autoradiography."}

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{"project":"craft-ca-core-dev","denotations":[{"id":"T2901","span":{"begin":13,"end":21},"obj":"CHEBI:60004"},{"id":"T2902","span":{"begin":46,"end":51},"obj":"CHEBI:46756"},{"id":"T2903","span":{"begin":52,"end":56},"obj":"CHEBI:32145"},{"id":"T2904","span":{"begin":73,"end":87},"obj":"CHEBI:32954"},{"id":"T2905","span":{"begin":95,"end":106},"obj":"CHEBI:62964"},{"id":"T2906","span":{"begin":113,"end":116},"obj":"CHEBI:18320"},{"id":"T2907","span":{"begin":141,"end":148},"obj":"CHEBI:27575"},{"id":"T2908","span":{"begin":157,"end":162},"obj":"CHEBI:3312"},{"id":"T2909","span":{"begin":175,"end":178},"obj":"CHEBI:37972"},{"id":"T2910","span":{"begin":233,"end":237},"obj":"PR:000010246"},{"id":"T2911","span":{"begin":233,"end":241},"obj":"GO:0097373"},{"id":"T2912","span":{"begin":367,"end":375},"obj":"CHEBI:60004"},{"id":"T2913","span":{"begin":480,"end":488},"obj":"CHEBI:75958"},{"id":"T2914","span":{"begin":507,"end":522},"obj":"CHEBI:26710"},{"id":"T2915","span":{"begin":527,"end":530},"obj":"CHEBI:8984"},{"id":"T2916","span":{"begin":667,"end":672},"obj":"CHEBI:3312"},{"id":"T2917","span":{"begin":771,"end":779},"obj":"MOP:0000780"},{"id":"T2918","span":{"begin":837,"end":841},"obj":"CHEBI:32145"},{"id":"T2919","span":{"begin":847,"end":854},"obj":"CHEBI:60004"},{"id":"T2920","span":{"begin":941,"end":955},"obj":"CHEBI:32954"},{"id":"T2921","span":{"begin":991,"end":997},"obj":"CHEBI:15882"},{"id":"T2922","span":{"begin":998,"end":1008},"obj":"CHEBI:35255"},{"id":"T2923","span":{"begin":1048,"end":1055},"obj":"CHEBI:16236"},{"id":"T2924","span":{"begin":1096,"end":1103},"obj":"CHEBI:16236"},{"id":"T2925","span":{"begin":1220,"end":1224},"obj":"CHEBI:16199"}],"text":"The reaction mixtures (25 μl) contained 25 mM HEPES–NaOH (pH 7.5), 50 mM sodium acetate, 10 mM Mg(CH3COO)2, 1 mM DTT, 0.25 mg/ml BSA, 0.5 mM ATP-γ-S, 0.5 mM CaCl2, 10 fmol of 32P-labeled bubble substrate, and the indicated amount of Mcm4/6/7 proteins. After incubation at 30°C for 30 min, the indicated amount of DNase I (TAKARA Biomedical, Japan) was added, and the mixtures were incubated at room temperature for 1 min. DNase I was inactivated by the addition of 250 μl of stop solution (20 mM EDTA, 0.2M sodium chloride, 1% SDS and 12.5 μg/ml yeast RNA). For nuclease P1 footprinting assay, binding reactions were carried out under the same conditions except that CaCl2 was omitted. P1 nuclease (0.3 U, Roche) was added and incubation was continued at 37°C for 1 min. Cleavage was stopped by addition of 50 μl of 25 mM EDTA and 0.4 M NaOH; the mixture was incubated at room temperature for 10 min followed by the addition of 12 μl of 3 M sodium acetate (pH 4.8). Proteins were removed by phenol–chloroform extraction, and DNAs were collected by ethanol precipitation followed by wash with 70% ethanol. The digested DNA was subjected to electrophoresis through a 10 or 12% polyacrylamide sequencing gel containing 7 M urea, followed by autoradiography."}