4.7.1 Nucleotides, DNA, and enzymes.
ATP, ADP, AMP, and Adenosine 5-(β,γ-imido)-triphosphate (AMP-PNP) were obtained from Sigma-Aldrich (St. Louis,MO); adenosine-5’-(γ-thio)-triphosphate (ATPγS) and dNTPs were from Roche (Basel, Switzerland). Labeled dNTPs and rNTPs were pur-chased from PerkinElmer (Boston, MA). Oligonucleotides used in this study were
syn-4.7. Supplemental Materials and Methods 73 thesized commercially by Integrated DNA Technologies (Coralville, IA). Single- stranded (ss) M13mp18 DNA was from Bayou Biolabs (Metairie, LA). The isolation of hPol!, hPol δ, RFC, PCNA, RPA and E. coli SSB were as previously described [133].
4.7.2 Antibodies.
Antibodies and their source were: Mcm subunits, Bethyl Laboratories (Montgomery, TX);
Cdc45, Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA); Psf1, Abgent (San Diego, CA); Sld5, Psf3, and Psf2 were prepared by Cocalico Biologicals, Inc. (Reamstown, PA) and affinity purified before use; Orc2, B.D. Pharmingen (San Diego, CA).
4.7.3 Construction of vectors for expression of hCMG.
cDNAs expressing each subunit of the Mcm2-7 and GINS complexes were PCR ampli-fied and subcloned into pFastBac1 (Invitrogen) for baculovirus expression of untagged proteins. Cdc45 and Sld5 cDNAs were cloned into pFastBacHtbFlag and pFastBacGST, respectively, for expression of N-terminally 6xHis-2xFLAG(HF) tagged Cdc45 and GST tagged Sld5 proteins.
4.7.4 Association of CMG with chromatin.
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics, and maintained at 37◦C in a humidified incubator containing 5% CO2. Chromatin fractionation experiments were performed as described [125] with modifications. Briefly, cells lysed in buffer containing 50 mM Hepes-NaOH/pH7.5, 100 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, 1 mM ATP, 50 mM sodium fluoride, 1 mM sodium vanadate and protease inhibitors were incubated at 4◦C for 10 min and the chromatin (C) and soluble fractions (SN) separated by centrifugation at 15,000 x g for 10 min. Chromatin pellets were resuspended in the above buffer, sonicated and treated with benzonase (Novagen, final concentration, 1.25 U/µl) at 40◦C for 2 h. Immunoprecipi-tation experiments were carried out by incubating 500µg of soluble or chromatin fractions with 200 ng of Cdc45 rabbit polyclonal antibodies, or non-specific GST rabbit polyclonal antibodies (Santa Cruz Biotechnology, Inc.), at 40◦C for 4 h, followed by the addition of 20µl of protein A agarose (Upstate Biotechnology) at 40◦C for 1 h. After centrifugation, beads were washed three times with above buffer, and bound proteins eluted with SDS loading buffer followed by SDS-PAGE separation on 4-20% gels. Immunoblot analysis was performed with rabbit polyclonal antibodies against Cdc45, Mcm2, Orc2 and Sld5 as indicated.
4.7.5 Isolation of hCMG Complex from 293 cells.
Cdc45 was subcloned into p3XFLAG-CMV to express N-terminally FLAG tagged Cdc45 and Sld5 was subcloned into pIRESpuro2. The two vectors were co-transfected into 293 human embryonic kidney cell and clones that stably-expressed FLAG-Cdc45 were selected. Over-expression of Sld5 was not stably maintained. Cells were grown in DMEM supplemented with 10% fetal bovine serum, antibiotics, and 2.5µg/ml puromycin. At 40% confluency, thymidine was added (final 2.5 mM) and cells were incubated for 18 hr to arrest cells in S phase. Sixty dishes were harvested (5 ml, wet volume), washed with ice-cold phosphate-buffered saline and frozen. Frozen cells were thawed, resuspended in 15 ml of extraction buffer (50 mM Hepes-NaOH/pH7.5, 5 mM MgCl2, 0.1% Triton X-100, 0.1 M NaCl, 1 mM ATP, 1X PhosSTOP (Roche) and protease inhibitors (1 mM PMSF, 1 mM benzamidine, 0.15 ˆIijM of aprotinin, 4 ˆIijM of leupeptin, and 1µM antipain) and the mixture centrifuged at 43,000 x g for 10 min at 4◦C. The supernatant was removed and the chromatin fraction resuspended in 15 ml extraction buffer and then sonicated. Chromatin was digested with Benzonase (15,000 unit) for 36 hr at 4◦C. Following digestion, the mixture was centrifuged at 43,000 x g for 45 min at 4◦C and the supernatant (chromatin extract) mixed with 0.4 ml Anti-FLAG M2 Affinity Gels pre-equilibrated with FW2 buffer (50 mM Tris-HCL/pH7.5, 0.2 M NaCl, 1 mM EDTA, and 1 mM DTT) for 3 hr on a rocking platform. The beads were collected by centrifugation at 290 x g for 5 min at 4◦C, washed 4 times with 10 ml FW2 buffer and bound proteins eluted 4 times with 0.3 ml FW2 buffer supplemented with 0.1 mg/ml 3xFLAG peptide. Eluates 1 and 2 were combined and an aliquot (0.5 ml) was layered onto a 5 ml 15-40% glycerol gradient (25 mM Tris-HCl/pH7.5, 0.1 M NaCl, 1 mM DTT, 1 mM EDTA) and centrifuged at 260,000 x g for 13.5 hr at 4◦C.
Fractions (150µl) were collected from the bottom of the tube.
4.7.6 Expression and purification of hCMG complex from insect cells.
Sf9 cells (2 x 106 cells/ml, 2 l) grown in suspension culture in Graceˆa ˘A´Zs medium sup-plemented with 10% fetal bovine serum at 27◦C, were infected with 11 viruses expressing Mcm2-7, HF-Cdc45, GST-Sld5, Psf3, Psf2, and Psf1. After 60 h at 27◦C, cells were har-vested by centrifugation at 650 x g for 5 min at 4◦C, washed once with ice-cold phosphate-buffered saline, and then frozen in dry ice and stored at −80◦C until used. The frozen pellet (20 ml) was thawed on ice and resuspended in 80 ml of hypotonic buffer (20 mM Hepes-NaOH/pH7.5, 5 mM KCl, 1.5 mM MgCl2, and protease inhibitors 1 mM PMSF, 1 mM benzamidine, 0.15µM of aprotinin, 4µM of leupeptin, and 1µM antipain). The cells were kept on ice for 10 min and then lyzed by Dounce homogenization (Pestle B, 30 strokes). The cell extract was adjusted to 0.42 M potassium acetate and centrifuged at 43,000 x g for 45 min at 4◦C. The cleared lysate was mixed with 2 ml anti-FLAG M2 Affin-ity Gels (Sigma-Aldrich) pre-equilibrated with FEQ buffer (20 mM Hepes-NaOH/pH7.5, 0.42 M potassium acetate, 5 mM KCl, 1.5 mM MgCl2) overnight on a rocking platform.
Following centrifugation at 290 x g for 5 min at 4◦C, the beads were washed 4 times with 50 ml of FW buffer (20 mM Hepes-NaOH/pH7.5, 0.42 M potassium acetate, 1 mM
4.7. Supplemental Materials and Methods 75 DTT, 1 mM EDTA, 0.01% NP-40, 10% glycerol, and protease inhibitors described above).
Bound proteins were eluted 5 times with 2 ml Q buffer (20 mM Hepes-NaOH/pH7.5, 1 mM DTT, 1 mM EDTA, 0.01% NP-40, 10% glycerol, and protease inhibitors) supplemented with 0.15 M potassium acetate and 0.1 mg/ml 3xFLAG peptide (Sigma-Aldrich). The eluted fractions were combined and applied to an 1 ml Q Sepharose Fast Flow column (1 ml, 1.5 x 1.5 cm, GE Healthcare Life Sciences) pre- equilibrated with Q buffer supple-mented with 0.15 M potassium acetate. Bound proteins were eluted with a linear gradient (14 ml) of potassium acetate (0.1-1 M) in Q buffer at a flow rate of 120µl/min and 360µl per fraction were collected. Fractions eluted between 0.54-0.94 M potassium acetate were combined and mixed with 0.3 ml Glutathione Sepharose 4B beads (GE Healthcare Life Sciences) pre-equilibrated with Prescission buffer (50 mM Tris-HCl/pH7.5, 0.15 M NaCl, 1 mM DTT, and 1 mM EDTA) overnight. The beads were washed 3 times with 10 ml Prescission buffer. The hCMG complex was eluted following cleavage of the GST tag from Sld5 by incubation of the beads with 368µl Prescission buffer plus 32µl (64 units) Prescission protease (GE Healthcare Life Sciences) for 6 hr at 4◦C. The eluted fraction (360µl) was layered on a 5 ml 15-40% glycerol gradient (25 mM Tris-HCl/pH7.5, 0.05 M NaCl, 1 mM DTT, 1 mM EDTA, and protease inhibitors) and centrifuged at 260,000 x g for 13.5 hr at 4◦C. Fractions (150µl) were collected from the bottom of the tube.
Fractions containing DNA helicase and ATPase activities that co-migrated with CMG complex were identified. This final step yielded between 12-35 pmol of hCMG complex (∼10 different preparations) distributed between 5 fractions. These fractions were stored at−20◦C or frozen at−80◦C after addition of 1 mg/ml BSA.
4.7.7 Expression and purification of hMcm4/6/7 complex from Sf9 cells.
For the purification of the Mcm4/6/7 complex, Mcm4 cDNA was PCR amplified and sub-cloned into pFastBacHtbFlag to express N-terminally 6xHis-2xFLAG(HF) tagged Mcm4.
Sf9 cells (2 x 106 cells/ml, 1 liter) were infected with viruses expressing HF- Mcm4, Mcm6, and Mcm7. After 60 hr of infection, cells were harvested and frozen as described for the pu-rification of the hCMG complex. The frozen pellet (8 ml) was thawed on ice, resuspended in 30 ml of hypotonic buffer and the lysate prepared as described above. The cleared lysate was mixed with 1 ml Anti-FLAG M2 Affinity Gels pre-equilibrated with FEQ buffer for 3 hr on a rocking platform. Following centrifugation at 290 x g for 5 min at 4◦C, the beads were washed 4 times with 50 ml FW buffer and bound proteins eluted 4 times with 1 ml Q buffer supplemented with 0.2 M potassium acetate and 0.1 mg/ml 3xFLAG peptide.
The eluted fractions were combined, applied to a Q Sepharose Fast Flow column (1 ml, 1.5×1.5 cm) pre-equilibrated with Q buffer supplemented with 0.15 M potassium acetate and bound proteins eluted with 10 ml linear gradient of potassium acetate (0.15-1 M) in Q buffer at a flow rate of 120µl/min and 360µl per fraction collected. Fractions contain-ing the Mcm4/6/7 complex were combined (total 2.8 ml) and concentrated to 0.5 ml by centrifugation (1200 x g) using the Centricon 50 (Millipore). The concentrated material (0.4 ml) was layered onto a 5 ml 15-40% glycerol gradient (25 mM Tris-HCl/pH7.5, 0.15 M potassium acetate, 1 mM DTT, 1 mM EDTA, and protease inhibitors) and centrifuged at
260,000 x g for 15 hr at 4◦C. Fractions (150µl) (collected from the bottom of the table) containing DNA helicase activity were frozen at−80◦C following the addition of 1 mg/ml BSA. This final step yielded 350 pmol of the Mcm4/6/7 complex. An SDS gel of the preparation, prior to addition of BSA, is shown in fig. 4.14.
4.7.8 Expression and purification of the hMcm2-7 complex from Sf9 cells.
For the purification of Mcm2-7, Mcm5 cDNA was PCR amplified and subcloned into pFastBacHtbFlag to express the N-terminally 6xHis-2xFLAG(HF) tagged Mcm5. Sf9 cells (2 x 106 cells/ml, 0.5 liter) were infected with 6 viruses expressing Mcm2-7 subunits including HF-Mcm5. After 60 hr of infection, cells were harvested and frozen as described above. The frozen pellet (4 ml) was thawed on ice, and the Mcm2-7 complex purified as described for the isolation of the Mcm4/6/7 complex. The final glycerol gradient step yielded 220 pmol of the Mcm2-7 complex. Active fractions (measured using the ATPase assay) were frozen at −80◦C after the addition of 1 mg/ml BSA. An SDS gel of the preparation prior to BSA addition, is shown in fig. 4.14.
4.7.9 Preparation of substrates for DNA helicase and EMSA assays.
The sequence of oligonucleotides used are listed in tab. 4.1. M13 DNA helicase substrates were prepared as follows; M13 complementary oligonucleotides (1 pmol) were 5’ 32P-labled with T4 polynucleotide kinase (New England Biolabs) and [γ-32P] ATP and annealed to M13 (1 pmol) in the presence of 0.15 M NaCl by heating for 3 min at 100◦C followed by slow cooling to 25◦C. Un-annealed oligonucleotides and unincorporated nucleotides were removed by Sepharose CL-4B column chromatography (GE Healthcare Life Sciences).
Substrates used to examine the processivity of the CMG helicase, M13-39-5’dT40 (tab. 4.1, oligonucleotide #3) were labeled, annealed to M13 and extended with SequenaseT M ver-sion 2.0 DNA polymerase (Affymetrix/USB corporation) [204]. Oligonucleotides used for DNA helicase and electrophoretic mobility shift assays (EMSA) were prepared as follows:
labeled oligonucletotides (10 pmol), were annealed to indicated oligonucleotides at molar ratios of 1:1 (or 1:1:1) and purified by 10% PAGE separation. The location of 32P in substrates is indicated by an asterisk.
4.7.10 EMSA assay.
Reactions (20µl) containing 25 mM Hepes-NaOH/pH7.5, 5 mM NaCl, 0.5 mM ATPγS, 10 mM magnesium acetate, 1 mM DTT, 0.1 mg/ml BSA, 2.5 fmol of DNA and indicated levels of protein were incubated at 37◦C for 30 min, followed by the addition of glycerol and bromphenol blue to 10% (v/v) and 0.05% (w/v), respectively. Products were subjected to 4% PAGE separation at 150 V in 1X TBE at 4◦C until the bromophenol blue migrated
4.8. Acknowledgments: 77