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{"project":"2_test","denotations":[{"id":"15917436-8390661-76775501","span":{"begin":427,"end":429},"obj":"8390661"},{"id":"15917436-12151393-76775502","span":{"begin":486,"end":488},"obj":"12151393"},{"id":"15917436-10567526-76775503","span":{"begin":853,"end":855},"obj":"10567526"},{"id":"15917436-12207017-76775504","span":{"begin":1306,"end":1308},"obj":"12207017"},{"id":"15917436-14609960-76775505","span":{"begin":2194,"end":2196},"obj":"14609960"},{"id":"15917436-14609960-76775506","span":{"begin":3325,"end":3327},"obj":"14609960"},{"id":"15917436-10567526-76775507","span":{"begin":3469,"end":3471},"obj":"10567526"}],"text":"MATERIALS AND METHODS\n\nReagents\nLabeled and unlabeled dNTPs/rNTPs and Sequenase were purchased from Amersham Pharmacia. M13mp18 and M13mp19 single-stranded circular DNA, T4 polynucleotide kinase, DNase I and the Klenow fragment were from TAKARA, Nuclease P1 was from Roche, and anti-FLAG M2 Ab (antibody)-agarose and FLAG peptide were from Sigma. SV40 T-antigen was purified from baculovirus-infected insect cells as reported (22). PriA helicase protein was provided by Dr Taku Tanaka (23). Oligonucleotides were purchased from Hokkaido system science Co., Ltd (Hokkaido, Japan), and were purified on polyacrylamide gel, followed by purification by Sep-Pak Plus C18 cartridge column (Waters).\n\nExpression and purification of mouse Mcm4/6/7 complex\nThe recombinant baculoviruses expressing His6-Mcm4/Mcm6 or Mcm7-FLAG proteins were previously described (11). Sf9 and High five insect cells were cultured at 27°C in Sf-900 II SFM (Life Technologies, Inc.) and EX-CELL 405 medium (JRH Biosciences), respectively. For expression of the Mcm4/6/7 proteins, High five cells were coinfected with recombinant baculoviruses expressing the His6-Mcm4/Mcm6 proteins and those expressing the Mcm7-FLAG, and were collected at 48 h post-infection. The recombinant Mcm4/6/7 complexes were purified as previously described (12).\n\nDNA substrates\nThe sequences for all the oligonucleotides used for constructions of DNA substrates are listed in Tables 1 and 2. The partial heteroduplex substrates were constructed by annealing labeled oligonucleotides with M13mp18 single-stranded circular DNA or its derivative. The labeled substrates were purified by Sepharose CL4B column chromatography (Amersham Pharmacia Biotech).\nThe DNA bubble substrates were assembled from two partially complementary oligonucleotides with top and bottom strand sequences (Table 1). T-tailed Y-fork substrates were prepared by annealing two oligonucleotides (dT30 and dT60 series). To construct the arrested fork and 3′- or 5′-extension substrates, various combinations of oligonucleotides shown in Table 1 were annealed. Bub-82/lamin B2 containing the T-rich strand of the lamin B2 origin region was constructed as previously described (13). The bubble substrate, c-myc/DUE or c-myc/DUE-C, contains sequences (GenBank accession number K01908) from nucleotides 722 to 853 or those from 828 to 747 (complementary strand), respectively, in the central melted region. For each substrate, labeled oligonucleotide (3 pmol) was annealed with the unlabeled oligonucleotide (6 pmol) in a reaction mixture (50 μl) containing 20 mM Tris–HCl (pH 7.5), 10 mM MgCl2 and 25 mM NaCl, which were heated to 95°C, kept at 67°C for 1 h, and then allowed to slowly cool down to 37°C. The assembled substrates were purified from polyacrylamide gel by elution into TE buffer (24).\nThe partial heteroduplex substrates on a single-stranded circular DNA with varied lengths of duplex regions were prepared by extending DNA chains from the 3′ end of the dT40-37mer (37mer region hybridizing at positions 6289–6326 of M13mp18 vector) annealed to the single-stranded circular DNA. DNA chains were elongated with Sequenase in the presence of [α-32P]dGTP, followed by extension after addition of ddGTP and all four dNTPs, resulting in substrates containing labeled duplex regions of varied lengths (13). The substrate (5 fmol) was first incubated with indicated amount of the Mcm4/6/7 complex at 37°C for 1 h in reaction mixture as described (11), and then reactions were stopped by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, followed by incubation for 20 min. Aliquots were electrophoresed on a 6.5% polyacrylamide gel in 1× TBE at 300 V for 2.5 h. The single-stranded circular DNAs used were M13mp18 and M13mp19+G-rich. The latter was constructed by inserting a G-rich repeat sequence (GGGGCGTGGGC)6, prepared by annealing of two oligonucleotides (5′-GATCC-[GGGGCGTGGGC]6-3′/5′-GATCC-[GCCCACGCCCC]6-3′) at the BamHI site of M13mp19 and the sequences of the insert were confirmed.\n\nDNA helicase and gel shift assays\nIn gel shift assays, Mcm4/6/7 proteins were incubated at 30°C for 30 min in reaction mixtures (15 μl) containing 25 mM HEPES–NaOH (pH 7.5), 50 mM sodium acetate, 10 mM Mg(CH3COO)2, 1 mM DTT, 0.25 μg/ml BSA, 0.5 mM ATP-γ-S and labeled substrates of the amount indicated. After addition of 2 μl of 50% glycerol, the reaction mixtures were directly applied on a polyacrylamide gel containing 6 mM magnesium acetate and 5% glycerol in 0.5× TBE, and electrophoresis was conducted at room temperature. For DNA helicase assays, the reaction mixtures of the gel shift assays, as described above, were incubated at 30°C for 15 min, and then the ATP was added at 10 mM, followed by incubation at 37°C for additional 30 min. The reactions were terminated by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, and were incubated for additional 15 min. The samples were separated by electrophoresis on nondenaturing polyacrylamide gel in 1× TBE.\n\nNuclease protection assays\nThe 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."}

PMC:1140370 / 4609-10893 JSONTXT

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PMC:1140370 / 4609-10893 JSON TXT