6704 Nucleic Acids Research, 2007, Vol. 35, No. 20 BrdU incorporation BrdU staining was performed with proliferating or diﬀerentiated C2C12 cells grown on coverslips. In principle, BrdU was added to cell media at 10 mM for 3 h. After ﬁxation with 70% ethanol (30 min), cells were incubated in 0.07 N NaOH for 2 min followed by a neutralization step with PBS, pH 8.5. Subsequently, cells were incubated with anti-BrdU (Becton Dickinson) in PBS containing 0.5% Tween 20 for 30 min. Immunoblotting analysis Extracts from proliferating and diﬀerentiating C2C12 cells were prepared by the following method. Cells were trypsinized and washed twice with PBS. Cells were lysed in cell lysis buﬀer (20 mM HEPES/NaOH, pH 7.9, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 10% glycerol, 0.2 mM EDTA, 1 PI-Mix, 1 mM DTT) for 15 min on ice. After centrifugation (2000 r.p.m., 5 min) the soluble supernatant was transferred to another tube and the pellet containing the nuclei was washed once (20 mM HEPES/NaOH, pH 7.9, 20% glycerol, 0.2 mM EDTA, 1 PI-Mix, 1 mM DTT). Nuclear proteins were extracted with high salt buﬀer (20 mM HEPES/NaOH, pH 7.9, 400 mM NaCl, 0.2 mM EDTA, 40% glycerol, 1 PI-Mix, 1 mM DTT) for 45 min on ice. Protein concentrations were determined by the method of Bradford (1976). Equal amounts (20 mg) of soluble and nuclear extract were subjected to SDS-PAGE and transferred onto nitrocellulose membrane (Schleicher & Schuell). The membrane was probed with primary antibodies followed by a secondary antibody conjugated with horseradish peroxidase and detected by the ECL system (Amersham Bioscience). RESULTS A Pre-RC-binding site is located within the origin of bidirectional replication of the mouse ribosomal gene cluster In a previous study, we have mapped an origin of bidirectional replication (OBR) in the murine rDNA locus by nascent strand determination analysis (Figure 1A) (32). Since replication is initiated within a region of 3 kb, we examined by ChIP assays whether ORC proteins are site-speciﬁcally assembled within this OBR. In asynchronously growing FM3A cells, proteins were cross-linked in vivo to DNA by formaldehyde and nucleoprotein complexes were immunoprecipitated with antibodies raised against ORC2 as well as with preimmune serum as a control. Precipitated DNA was ampliﬁed via PCR using primer pairs each ﬂanking 1 kb of the OBR. A sequence-speciﬁc association of ORC2 proteins was assumed if the signals of PCR analyses obtained by speciﬁc antibodies exceeded those by preimmune sera. In ﬁrst experiments, ORC2 was found cross-linked with DNA sequences located 3 to 2 kb upstream of the transcription start site (data not shown). To further conﬁne this binding site, three subfragments of this region were used in subsequent ChIP experiments (Figure 1A, fragments A: 2837 to 2499, B: 2519 to 2152 and C: 2255 to 1820). PCR analysis following immunoprecipitation with antibodies against ORC2 pro- duced a strong signal with primer pairs for fragment B, whereas intensities of PCR products A or C were signi- ﬁcantly weaker (Figure 1B). This suggests that a pre-RC binding site is located within fragment B, situated 2519 to 2152 upstream the transcription start site. As an additional negative control, PCR analyses were performed with primers speciﬁc for a region in the 5.8 S rRNA gene, fragment D. ORC2 was not to be found interacting with this DNA sequence in the coding region. Cell cycle-dependent binding of pre-RC proteins in murine FM3A cells To study the binding of initiator proteins to fragment B in the murine rDNA locus during the cell cycle, ChIP assays with synchronized FM3A cells were performed. FM3A cells were arrested in G1 phase, at the G1/S transition and in S phase as described in Materials and Methods section. Cross-linked chromatin was immunoprecipitated with antibodies against ORC1, -2, -4 and -5, respectively, as well as with preimmune sera. During G1 phase and at G1/S transition ORC1, -2, -4 and -5 are bound to fragment B (Figure 2, upper and middle panel). During S phase, ORC1 dissociates from the pre-RC-binding site (Figure 2, lower panel). Densitometric values of PCR results are shown in Supplementary Figure 1. Apparently, fragment B has an intrinsic, unspeciﬁc protein-binding capacity resulting in a higher intensity of PCR bands with control antibodies and primer pair B compared to primer pairs A, C or D. Isolated pre-RC proteins associate with origin DNA in vitro In order to further verify the speciﬁcity of the pre- RC-binding site at fragment B, we performed EMSAs with partially puriﬁed nuclear extract and the DNA fragments described above. Proteins from FM3A nuclear extracts were fractionated by three successive chro- matographic steps as described in Materials and Methods section. To analyze the DNA-binding activity, puriﬁed proteins of >300 kDa mass after gel ﬁltration were incubated with radiolabeled fragment B and assayed by EMSA (Figure 3A). Proteins of several fractions bind to DNA fragment B, forming two major protein/DNA complexes of distinct electrophoretic mobility, designated complexes A and B, respectively. The fractions exerting the most prominent shifts, i.e. E5 (molecular mass of >663 kDa) and E11 (<440 kDa), were examined by immunoblotting using various antibodies speciﬁc for components of the pre-RC (Figure 3B). ORC1, -2, -4 and -5 were conﬁrmed in fraction E5. Fraction E11 contained the same proteins but at a lower level than in fraction E5. By this three-step chromatography, ORC containing protein complexes could be enriched from nuclear extracts. Fractions E5 and E11 comprised highest DNA-binding activity resulting in the formation of complex A and B, respectively. Although the presence of pre-RC compo- nents was demonstrated in these protein fractions, the data could not provide conclusive evidence whether the complexes are caused primarily by binding of pre-RC