The hCMG complex is associated with chromatin. Soluble (SN) or chromatin (C) fractions (500 μg) isolated from HeLa cells were incubated with Cdc45 antibodies (lane 3 and 4) or nonspecific (GST) antibodies (lane 5 and 6), as indicated at the top of the immunoblots; specific interactions were detected by Western blotting using antibodies to Mcm2, Cdc45, Sld5, or Orc2. Input represents 10% of the lysate used for immunoprecipitation (lanes 1 and 2). The IgG bands in the Cdc45 immunoblot (lanes 3–6) are indicated by an asterisk.

Purification of hCMG complex. (A) Silver-stained gel of glycerol gradient fractions of purified hCMG complex. Each gradient fraction (15 μL) was loaded onto a 4–20% gel (Invitrogen). The position of standard proteins (GE Healthcare Life Sciences) following sedimentation is indicated above the fractions (669 kDa, thyroglobulin; 150 kDa, aldolase; and 75 kDa, conalbumin). (B) Enlargement of the silver-stained fraction 7 shown in A. Protein bands are labeled at the right of the gel. (C) Cosedimentation of DNA helicase activity with the hCMG complex. Helicase assays were performed with aliquots (0.5 μL) of each fraction as described in . The structure of the helicase substrate containing a 39-mer duplex region and a 5′-dT₄₀ (M13 annealed to labeled oligonucleotide 3) is shown on the left side of the gel. B, boiled substrate.

DNA helicase activity of the hCMG complex on various DNA substrates. (A) Increasing levels of CMG (7.5, 15, and 30 fmol) were incubated with oligonucleotide substrates (see Table S1 for description of oligonucleotides used in this assay). The first two substrates (I and II; Upper) were formed by annealing oligonucleotide 3 (labeled) with 9 or 11, respectively. Substrate III was prepared by annealing oligonucleotide 3 (labeled) with 11 (bottom strand) and 14. Substrate IV was made by annealing oligonucleotide 15 (labeled) with 5 and 9 (bottom strand). The three substrates (V, VI, and VII; Lower) were synthesized by annealing oligonucleotide 3 (labeled) with oligonucleotide 12, 13, or 10, respectively. Substrate VIII was made by annealing oligonucleotide 3 (labeled) with oligonucleotides 12 (bottom strand) and 14. Substrate IX was generated by annealing oligonucleotide 14 (labeled) with oligonucleotides 12 (bottom strand) and 3. (B) Helicase assays with oligonucleotide substrates containing different 3′-tail lengths. Substrates containing the 3′-oligo(dT₄₀) or 3′-oligo(dT₈₀) tails are the same as I and VII described in A. Substrates containing 3′-oligo(dT₂₀) tails was made by annealing oligonucleotide 3 (labeled) with 8. CMG (15 fmol) was incubated with substrates for varying incubation periods, and the substrate unwound (%) plotted against the time of incubation. (C) Helicase assays with M13 substrates containing a 39-mer duplex region and different 5′-dT tail lengths (0, 20, 40, and 60 nt) prepared by annealing labeled oligonucleotides 1, 2, 3, or 4 with M13. The unwound substrate formed (%) was calculated and plotted against the level of CMG complex added.

Processivity of the CMG helicase activity. (A) Substrates used for the processivity studies were prepared as described in . For the preparation of the short duplex DNA substrate (39–500 bp) used in lanes 1–5, the annealed oligonucleotide was extended in the presence of dNTPs containing ddCTP. For the preparation of the long duplex substrate (>500 bp), described in lanes 6–10, the annealed oligonucleotide was extended in the absence of ddCTP. Increasing levels of CMG (12.5, 25, and 50 fmol) were incubated with DNA substrates as described in . (B) Comparison of the processivity in the presence and absence of RPA or E. coli SSB. Substrates containing duplex regions, as shown, were preincubated with 40 fmol the hCMG complex in the presence of 0.05 mM ATP and then supplemented with 0.45 mM ATP and RPA (1.7 pmol) or E. coli SSB (0.27 pmol) followed by further incubation for 30 min.

DNA binding activity of the hCMG complex. EMSA assays were performed with DNA substrates under different conditions. Reactions contained 15 fmol of hCMG. (A) EMSA assays with fork-structured DNAs containing 5′- or 3′-oligo(dT₄₀) tails prepared by annealing oligonucleotides 3 and 9 (Table S1). (Right) EMSA assays were performed with the nucleotides indicated at the top of the gel. The DNA products formed (substrate–protein complex, substrate, and unwound product) are illustrated. (B) EMSA assays with substrates shown at top of gels. The first three substrates used were prepared by annealing oligonucleotides 1 with 7, 3 with 7, and 1 with 9. The next two substrates were prepared with labeled oligonucleotides 3 and 9, and the last substrate indicated was identical to that described in A. (C) CMG binding to fork-structured substrates containing different 3′-tail lengths. The substrates used were the same as those described in Right. The nucleoprotein complex formed (%) was calculated and plotted against the CMG complex added.

Rolling circle assay. The primed 200-nt minicircle was synthesized as described in SI Materials and Methods. (A) Requirements for Pol ε-catalyzed leading-strand synthesis. Reactions (15 μL) containing 20 mM Tris⋅HCl (pH 7.5), 10 mM magnesium acetate, 10 mM potassium glutamate, 1 mM DTT, 0.1 mg/mL BSA, 0.2 mM EDTA, 3.75% glycerol, 0.5 mM AMP-PNP, rolling circle DNA substrate (50 fmol), and hCMG complex (15 fmol) were incubated for 10 min at 37 °C; 0.12 mM dCTP, 0.12 mM, dGTP, and 0.03 mM [α-³²P]dATP (specific activity 37,700 cpm/pmol) were added with RFC (20 fmol), PCNA (1 pmol), and hPol ε (70 fmol). After 5 min at 37 °C, 5 mM ATP was added and the reaction incubated for 10 min, after which E. coli SSB (0.5 pmol) was added and the mixture incubated for 60 min. Mixtures were adjusted to 10 mM EDTA and separated on an alkaline agarose gel (1%) at 15 W for 2.5 h. The gel was washed with water, dried, and audioradiographed at −80 °C. (B) Comparison of leading-strand synthesis by hPol ε and hPol δ. Reactions were as described in A with 50 mM potassium glutamate, 50 fmol CMG complex, and 35 and 70 fmol hPol ε or 35, 70, and 300 fmol of hPol δ, where indicated. After incubation, samples were treated with Proteinase K (0.1 mg/mL) in reactions containing 20 mM EDTA, 1% SDS, and 40 μg of yeast tRNA. Following ethanol precipitation, samples were subjected to alkaline agarose gel separation as described above. (C) Elongation of singly primed M13 by hPol ε and hPol δ. The activity of hPol ε (55 fmol) and hPol δ (44 fmol) observed with singly primed M13 (7 fmol) following incubation for 30 min at 37 °C was carried out as previously described ().