was quenched by adding glycine to a ﬁnal concentration of 0.125 M. Cells were washed in PBS including protease inhibitors and lysed by incubation in cell lysis buﬀer (10 mM Tris, pH 8.0, 10 mM NaCl, 0.2% NP-40, 0.5 mM PMSF, 1 PI-Mix: 2 mg/ml pepstatin, 2 mg/ml leupeptin, 5 mg/ml aprotinin) for 10 min at 48C, followed by centrifugation at 600g for 5 min. Nuclei were lysed in nuclei lysis buﬀer (50 mM Tris, pH 8.1, 10 mM EDTA, 1% SDS, 0.5 mM PMSF, 1 PI-Mix) and incubated with acid-washed glass beads for 40 min on a shaker. The extract was sonicated 7 for 30 s each time and centrifuged. The supernatant containing chromatin was used for chromatin immunoprecipitation. Chromatin immunoprecipitation assay DNA fragments of less than 500 bp were obtained by incubation with 45 U micrococcal nuclease S7 (Roche) per 200 mg nucleoprotein for 15 min at 378C in the presence of 20 mM CaCl2. Reactions were stopped by adding EDTA (20 mM ﬁnal). The extract was divided into three equal volumes. One aliquot was removed and used in the PCR analysis as input control. The remainder of chromatin was diluted with IP dilution buﬀer (20 mM Tris, pH 8.1, 150 mM NaCl, 3 mM EDTA, 0.01% SDS, 1% Triton X-100) and incubated overnight at 48C with 10–15 mg antibodies or preimmune sera in a ﬁnal volume of 800 ml. Thirty microliter of protein A-Sepharose were added and samples were incubated for 4 h at 48C. Each batch of resin was washed 5 with wash buﬀer I (20 mM Tris, pH 8.1, 150 mM NaCl, 2 mM EDTA, 0.1% SDS 1% NP-40), 3 with wash buﬀer II (10 mM Tris pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% Na-deoxycholic-acid) and 3 with TE (10 mM Tris, pH 8.0, 1 mM EDTA). DNA–protein complexes were eluted twice with 100 ml elution buﬀer (100 mM NaHCO3, 1% SDS) for 10 min at 378C. After RNase A treatment (0.06 mg/ml) in the presence of NaCl (0.3 M) for 30 min at 378C, cross-links were reverted for 30 min at 658C. DNA was recovered by treating the samples overnight at 378C with 0.2 mg/ml proteinase K, followed by two extractions with phenol/chloroform and ethanol precipitation. DNA was resuspended in 45 ml bdH2O and analyzed by PCR with primer pairs speciﬁc for fragment A, B, C and D, respectively. The ampliﬁcation program consisted of 2 min at 948C, followed by 32 cycles of 30 s at 948C, 30 s at 528C, 30 s at 728C and ﬁnally 7 min at 728C. PCR band intensities were analyzed by Aida v.3.5. A sequence- speciﬁc association of pre-RC proteins was assumed if the PCR signals obtained by speciﬁc antibodies were stronger than those by preimmune sera. Preparation of nuclear extracts and protein purification by cation exchange and gel filtration chromatography FM3A cells were washed in PBS and resuspended in hypo- tonic lysis buﬀer (10 mM HEPES, pH 7.8, 10 mM NaCl, 1.5 mM MgCl2, 5 mM b-mercaptoethanol, 1 PI-Mix, 1 mM DTT). After the cell suspension was homoge- nized in a Dounce homogenizer, 1/10 volume of 10 salt buﬀer (300 mM HEPES, pH 7.8, 1400 mM KCl, 30 mM MgCl2, 5 mM b-mercaptoethanol, 1 PI-Mix, Nucleic Acids Research, 2007, Vol. 35, No. 20 6703 1 mM DTT) was added. After centrifugation (24 000g, 30 min) the pellet containing the nuclei was resuspended in KEP buﬀer (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM KCl, 0.2 mM EDTA, 1.5 mM MgCl2, 5 mM b-mercaptoethanol, 1 PI-Mix, 1 mM DTT) and rotated for 1 h at 48C. After centrifugation (12 000g, 30 min) the nuclear extract was dialyzed overnight against buﬀer H (50 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM EGTA, 5 mM Na-acetate, 10% glycerol, 5 mM b-mercaptoetha- nol, 1 PI-Mix, 1 mM DTT) containing 250 mM NaCl. The dialyzed sample was ﬁrst puriﬁed on a 5 ml cation exchange column (HiTrap Heparin HP, Amersham Biosciences) and bound proteins were eluted with a linear gradient of NaCl (250–800 mM). Samples were analyzed on immunoblots for the presence of ORC2. Fractions containing ORC2 were combined and dialyzed overnight against buﬀer H containing 100 mM NaCl. The extract was further puriﬁed by chromatography over a 1 ml cation exchange column (HiTrap SP Sepharose Fast Flow, Amersham Biosciences) and bound proteins were eluted with a linear gradient of NaCl (100– 500 mM). After immunoblot analysis using antibodies against ORC1 and -2 fractions containing ORC1 and ORC2 were concentrated on Centrifugal Filter Devices (Amicon Ultra, Millipore) and subjected to gel ﬁltration on a Superdex 200 PC 3.2/30 column (Amersham Biosciences). Electrophoretic mobility shift assay Oligonucleotides were labeled with g32P-ATP using T4 polynucleotide kinase. PCR reactions were carried out as described above. EMSAs (10 ml) contained 10 mM HEPES, pH 7.5, 5 mM MgCl2, 1 mM CaCl2, 70 mM KCl, 250 ng poly dI+dC, 5 mM ATP (as indicated), 10 fmol g32P-ATP 50 end-labeled DNA fragments and 400 ng puriﬁed nuclear extract. Binding reactions were incubated for 45 min on ice, 10 min at 308C, and again 45 min on ice. For supershift assays, 100 ng of either speciﬁc or control antibodies were added for a further 2 h on ice. Electrophoresis was performed on 4% native polyacrylamide gels at 100 V in 0.5 TBE and at 48C. Following electrophoresis, gels were dried and autoradiographed. Site-directed mutagenesis The PCR mixture contained 20 ng template plasmid DNA (1545 bp rDNA fragment from 3476 to 1931 cloned into pUC18), 15 pmol of each oligonucleotide, 200 mM dNTPs, 2.5 U Pfu DNA polymerase (Stratagene) and buﬀer in a total volume of 50 ml. PCR was performed under the following conditions: denaturation at 958C for 30 s, followed by 18 cycles of 958C for 30 s, 558C for 1 min and 728C for 1.5 min/kb. The reaction was incubated with 10 U of DpnI for 1 h at 378C to digest methylated, non-mutated parental DNA. An aliquot (1/5) of the PCR product was transformed in 100 ml of competent JM109 Escherichia coli cells (Stratagene). The presence of the mutation was conﬁrmed by DNA sequencing of the constructs.