PMC

Orc2p and Mcm proteins on crosslinked chromatin. (A) Immunoprecipitations with antibodies against Orc2p (α-Orc2) and with non-specific control antibodies (IgG). Input (crosslinked, nuclease-treated chromatin before immunoprecipitation), supernatants (remaining chromatin after treatment with antibodies and protein A–Sepharose) and immunoprecipitates were analyzed in western blots with Mcm3p-specific (MCM3) and with Orc2p-specific antibodies (ORC2). (B) Immunoprecipitations with antibodies against Mcm3p (α-MCM3) and with non-specific control antibodies (IgG). The input, supernatant and precipitated samples were western blotted and analyzed with antibodies against Mcm3p (MCM3), Mcm4p (MCM4), Mcm5p (MCM5) and Mcm7p (MCM7) as well as with antibodies against Orc2p (ORC2). (C) Immunoprecipitation with antibodies against Mcm4p (α-MCM4) and with non-specific control antibodies (IgG). The input, supernatant and precipitated samples were western blotted and analyzed with Mcm4p, Mcm6p and Mcm7p antibodies as well as with antibodies against Orc2p as indicated.

ChIP assay on crosslinked chromatin from synchronized HeLa cells. Crosslinked chromatin from G₁ phase cells and S phase cells was immunoprecipitated with antibodies against Mcm3p (α-MCM3). The input, supernatant and precipitated samples were western blotted and analyzed with Mcm3p, Mcm5p and Orc2p antibodies as well as with antibodies against the large subunit of RPA (RPA70) as indicated. Synchronization was verified by analysis of DNA content on a flow cytometer (shown in the conventional manner).

Specific DNA regions in immunoprecipitated crosslinked chromatin from cells in G₁ phase and S phase. HeLa cells were synchronized in G₁ phase (left) or released into S phase for 4 h (right) and were subjected to ChIP analysis. Quantitative real-time PCR was performed with DNA templates extracted from chromatin precipitated with antibodies against (A) Orc2p, (B) Mcm3p and (C) Mcm4p. We show the results of three independent experiments. (Below) Schematic representation of the genomic region analyzed with the PCR-amplified segments indicated by arrowheads.

Enrichment of specific DNA sequences in immunoprecipitates. Quantitative PCR gives the results in ‘genomic units’ relative to serially diluted purified genomic DNA. The ratios of precipitated over input genomic units are multiplied by 100 and plotted against the primer sites on the analyzed region. The results of the precipitations with Orc2 antibodies are summarized in (A), with Mcm3 antibodies in (B) and with Mcm4 antibodies in (C). Open squares indicate the mean enrichment of the G₁ phase samples and closed circles indicate the mean enrichment of the samples from S phase cells.

(Opposite) Specific DNA regions in immunoprecipitated crosslinked chromatin from unsynchronized HeLa cells. (A) Genomic organization of the analyzed region encompassing human genes PRKDC and MCM4 (). 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, divergent arrowheads show regions complementary to the PCR primers used. (B) Input. DNA was extracted from crosslinked chromatin before immunoprecipitation and amplified by quantitative PCR using the primer sets indicated. The results are expressed in ‘genomic units’ relative to serially diluted genomic control DNA. (C) Immunoprecipitated chromatin. Quantitative real-time PCR with DNA templates extracted from chromatin precipitated with control antibodies and with antibodies against Orc2p, Mcm3p and Mcm4p as indicated.