DNA substrates
The 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).
The 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).
The 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.