Cross-linked chromatin is efficiently immunoprecipitated. (A) DNA in cross-linked nucleoprotein before (−) and after (+) treatment with micrococcal nuclease (MNase). (B) Immunoprecipitation of cross-linked, micrococcal nuclease-treated chromatin with antibodies against hMcm3p (α-MCM3) and with unspecific control antibodies (IgG). Input (chromatin sample before immunoprecipitation) and supernatants (remaining chromatin after treatment with antibodies and protein A-Sepharose) are analyzed in Western blots with α-MCM3. Immunoprecipitates are analyzed with DEFD antibodies that recognize a polypeptide common to all human Mcm proteins (49) and with hOrc2p-specific antibodies. (C) Immunoprecipitation of cross-linked, micrococcal nuclease-treated chromatin with hOrc2p-specific (α-ORC2) and hOrc1p-specific (α-ORC1) antibodies as indicated. The input, supernatant, and precipitated samples were Western blotted and analyzed with hOrc1p and hOrc2p antibodies, as well as with antibodies against a large subunit of RNA polymerase II.

Quantitative PCR. Broken lines, two dilution series of genomic HeLa cell DNA. Dilutions are based on the estimate that 30 ng of DNA correspond to 10⁴ genome equivalents. The x axis as calculated by the Light Cycler software cuts parallel running calibration curves. The y axis corresponds to fluorescence in arbitrary units. Solid lines show template DNA extracted from immunoprecipitated chromatin (gray line, Orc1 precipitated; black line, Orc2 precipitated). (A) PCR primers corresponding to the UPR (see Fig. 3C and Table 1). (B) PCR primers corresponding to MCM4 exon VII (Table 1).

S-phase chromatin contains no hOrc1p. (A) Cross-linked chromatin from G₁-phase cells and S-phase cells was immunoprecipitated with hOrc1p antibodies (α-ORC1) or hOrc2p antibodies (α-ORC2). Input (I), supernatants (S), and precipitates (P) of the α-ORC1- and α-ORC2-treated samples were analyzed with hOrc1 and with hOrc2p antibodies on Western blots. (B) Schematic representation of the genomic region analyzed in this study. The positions of the segments amplified by PCR are indicated by black boxes. (C) DNA extracted from chromatin immunoprecipitates of G₁-phase cells (upper panels) and of S-phase cells (lower panels) were PCR amplified as in Fig. 5. We show the results of two independent experiments.

Sequences of the replication start site of the lamin B2 origin are enriched in the ORC immunoprecipitate. (A) Quantitative real-time PCR was used to determine the copy numbers of sequences in cross-linked, nuclease-treated input chromatin. (B) The input chromatin sample was immunoprecipitated with hOrc2p-specific antibodies (α-ORC2 ppt). The copy numbers of amplifiable sequences were determined in the precipitates. Also shown are control immunoprecipitation experiments performed under identical conditions with nonspecific antibodies (control IgG). (C) Enrichment of sequences expressed as the ratio of precipitated over input DNA.

Short treatment with formaldehyde suffices to cross-link Orc proteins to DNA. HeLa cells were treated with formaldehyde for 2 to 8 min as indicated. Nucleoprotein was prepared by repeated wash steps and CsCl purification (Materials and Methods). Aliquots (100 μg of DNA each) were suspended in Laemmli buffer and analyzed by denaturing polyacrylamide gel electrophoresis (32). We show Coomassie blue-stained gels and immunoblots (Western blot) (60) with polyclonal monospecific antibodies against human proteins Mcm3p and Mcm5p (49), as well as hOrc1p and hOrc2p (30) as indicated. The asterisk indicates a degradation product of hOrc1p.

hOrc2p is specifically associated with the UPR of gene MCM4. (A) Genomic organization. In the double-line diagram, exons are shown as black boxes, and arrows indicate the starts and directions of transcription. In the single-line diagram, boxes show regions amplified in PCRs, and the vertical lines show NciI restriction sites. (B) PCR assays. UPR, primers complementary to the MCM4 upstream region; Exon 9, primers complementary to exon IX; HeLa DNA, PCR assays with genomic DNA as template (+) or without template DNA (−); α-ORC2 ppt, template DNA extracted from cross-linked chromatin immunoprecipitated with anti hOrc2p antibodies; control ppt, DNA extracted from mock immunoprecipitates with control antibodies. (C) DNA in cross-linked material before (−) and after (+) digestion with NciI. (D) Intergenic region. The translational start codons of the two divergent genes are marked by gray horizontal arrows. Multiple transcriptional start sites (black horizontal arrows with numbers) have been mapped by Conelly et al. (11) for the gene PRKDC and for the gene MCM4. Short vertical arrows indicate NciI restriction sites. Boxed sequences show complementary to PCR primers. Gray backgrounds indicate portions of the Alu-Jo repetitive element and the central AT-rich sequence.

hOrc1p- and hOrc2p-bearing MCM4 upstream promoter DNA is enriched in chromatin immunoprecipitates. (A) Overview over the genomic region encompassing genes PRKDC and MCM4. Black boxes, exons; gray boxes, repeat elements; divergent arrow heads, sequences complementary to the PCR primers used (see Table 1). (B) Quantitative real-time PCR with DNA templates extracted from chromatin precipitated with control antibodies and with antibodies against hOrc1p and hOrc2p. We show the results of two independent experiments.

The UPR of gene MCM4 is highly abundant among nascent DNA strands. (A) Rationale and procedure (see the text). (B) In the left panel, quantitation of the DNA sequences in the 1- to 2-kbp fraction of nascent DNA is shown. The Primers UPR, In7, In6, In1, Ex2, Ex6, Ex7, and Ex9 (Table 1) were used in two independent PCRs (solid and broken line). In the right panel, LB2 corresponds to a primer pair with sequences of the known and well characterized lamin B2 replication origin (23). LB2C1 is a control primer pair complementary to a genomic region in a 3.5-kbp distance upstream from the lamin B2 origin (see Table 1).