The Origin Recognition Complex Marks a Replication Origin in the Human TOP1 Gene Promoter
2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês
10.1074/jbc.m202165200
ISSN1083-351X
AutoresChristian Keller, Eva‐Maria Ladenburger, Marcel Kremer, Rolf Knippers,
Tópico(s)Chromosomal and Genetic Variations
ResumoThe locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the humanTOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation. The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the humanTOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation. autonomously replicating sequences origin recognition complex chromatin immunoprecipitation matrix attachment regions scaffold attachment factor A Origins of DNA replication are the chromosomal regions where DNA replication forks for bidirectional duplication of replicons are established. The large and discontinuous genomes of eukaryotes require a large number of origins that are distributed throughout the genome to guarantee a complete replication within the limited time of the S phase in a cell division cycle (for reviews, see Refs. 1Bielinsky A.K. Gerbi S.A. Mol. Cell. 1999; 3: 477-486Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 2Boulikas T. J. Cell. Biochem. 1996; 60: 297-316Crossref PubMed Scopus (55) Google Scholar, 3DePamphilis M.L. 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Raghuraman M.K. Fangman W.L. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 425-434Crossref PubMed Scopus (23) Google Scholar, 11Newlon C.S. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Plainview, NY1996: 873-914Google Scholar). Prototypic budding yeast origin (autonomously replicating sequences (ARSs)1) are composed of 100–200 base pairs and contain several essential sequence elements including a domain A with the AT-rich ARS consensus sequence and three short stimulatory elements, B1-B3, which are functionally important but divergent in sequence (12Marahrens Y. Stillman B. Science. 1992; 255: 817-823Crossref PubMed Scopus (485) Google Scholar). The ARS consensus sequence and the adjacent B1 domain element constitute a binding site for proteins of the origin recognition complex (ORC), whereas the B3 domain element forms a binding site for the transcription factor Abf1 in some, but not all yeast origins (13Diffley J.F. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2120-2124Crossref PubMed Scopus (157) Google Scholar). ORC is a multimeric protein complex composed of six essential subunits (Orc1p-Orc6p) that associate in an ATP-dependent manner with ARSs (13Diffley J.F. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2120-2124Crossref PubMed Scopus (157) Google Scholar, 14Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (992) Google Scholar, 15Lee D.G. Bell S.P. Mol. Cell. Biol. 1997; 17: 7159-7168Crossref PubMed Scopus (166) Google Scholar). The major known function of ORC appears to be the recruitment of factors such as Cdc6, Mcm proteins, and others for the formation of functional pre-replication complexes (16Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 17Diffley J.F. Cocker J.H. Dowell S.J. Rowley A. Cell. 1994; 78: 303-316Abstract Full Text PDF PubMed Scopus (468) Google Scholar, 18Kelly T.J. 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Biol. 1995; 15: 2482-2489Crossref PubMed Scopus (59) Google Scholar) were established to determine the sites where bidirectional genome replication initiates. Using these experimental strategies, a small number of mammalian origins have been identified. One major conclusion of these experiments is that, whereas the replication of genomes in differentiated mammalian and other metazoan cells begins at specific genomic loci that are quite stably inherited from one cell division cycle to the next, individual origins of a given organism differ greatly in size and sequence and are clearly less uniform and more complex in structure than budding yeast origins (3DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Plainview, NY1996: 31Google Scholar, 4DePamphilis M.L. Bioessays. 1999; 21: 5-16Crossref PubMed Scopus (174) Google Scholar, 5Coverley D. Laskey R.A. Annu. Rev. Biochem. 1994; 63: 745-776Crossref PubMed Scopus (160) Google Scholar, 9Todorovic V. Falaschi A. Giacca M. Front. Biosci. 1999; 4: 859-868Crossref PubMed Google Scholar, 10Brewer B.J. Diller J.D. Friedman K.L. Kolor K.M. Raghuraman M.K. Fangman W.L. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 425-434Crossref PubMed Scopus (23) Google Scholar, 24Berezney R. Dubey D.D. Huberman J.A. Chromosoma (Berl.). 2000; 108: 471-484Crossref PubMed Scopus (309) Google Scholar). Many known mammalian origins are found to be located between transcribed regions and frequently in the vicinity of active transcriptional start sites (25Araujo F.D. Knox J.D. Ramchandani S. Pelletier R. Bigey P. Price G. Szyf M. Zannis-Hadjopoulos M. J. Biol. Chem. 1999; 274: 9335-9341Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 26Iguchi-Ariga S.M. Okazaki T. Itani T. Ogata M. Sato Y. Ariga H. EMBO J. 1988; 7: 3135-3142Crossref PubMed Scopus (105) Google Scholar, 27Little R.D. Platt T.H. Schildkraut C.L. Mol. Cell. 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Falaschi A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1498-1503Crossref PubMed Scopus (51) Google Scholar). A Drosophila melanogaster ORC localizes in vivoto the chorion gene amplification control element, which is active in ovarian follicle cells and determines the amplification of chorion gene clusters by repeatedly initiating DNA replication (32Austin R.J. Orr-Weaver T.L. Bell S.P. Genes Dev. 1999; 13: 2639-2649Crossref PubMed Scopus (173) Google Scholar). Interestingly, amplification control element-bound ORC is in close contact with transcription factor E2F, which together with the Rb protein, regulates the initiation of replication (33Bosco G., Du, W. Orr-Weaver T.L. Nat. Cell Biol. 2001; 3: 289-295Crossref PubMed Scopus (203) Google Scholar). An ORC binding site has also been identified in the Epstein-Barr virus genome. The 165-kbp viral chromosome replicates as an episome in latently infected human cells in a regulated once-per-cell cycle manner dependent upon a functional bipartite viral origin. This origin binds the viral initiator protein, EBNA-1, in addition to proteins of the human ORC, which appear to be essential for viral genome replication (34Chaudhuri B., Xu, H. Todorov I. Dutta A. Yates J.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10085-10089Crossref PubMed Scopus (180) Google Scholar, 35Schepers A. Ritzi M. Bousset K. Kremmer E. Yates J.L. Harwood J. Diffley J.F. Hammerschmidt W. EMBO J. 2001; 20: 4588-4602Crossref PubMed Scopus (190) Google Scholar). Using a modified version of the chromatin immunoprecipitation (ChIP) protocol in combination with quantitative real-time PCR, we have recently identified an ORC binding site between two divergently transcribed human genes in a region that coincides with a start site for bidirectional DNA synthesis (36Ladenburger E.-M. Keller C. Knippers R. Mol. Cell. Biol. 2002; 22: 1036-1048Crossref PubMed Scopus (130) Google Scholar). We have now used the ChIP technique to investigate another transcription unit in the human genome, the TOP1 gene, which occupies ∼100 kbp of the chromosome 20 sequence (37Kunze N. Yang G.C. Dolberg M. Sundarp R. Knippers R. Richter A. J. Biol. Chem. 1991; 266: 9610-9616Abstract Full Text PDF PubMed Google Scholar). The TOP1 gene promoter co-localizes with a CpG island and contains an A+T-rich element (38Kunze N. Klein M. Richter A. Knippers R. Eur. J. Biochem. 1990; 194: 323-330Crossref PubMed Scopus (39) Google Scholar). In addition, the gene has two well characterized matrix attachment regions (MARs; Ref. 39Romig H. Ruff J. Fackelmayer F.O. Patil M.S. Richter A. Eur. J. Biochem. 1994; 221: 411-419Crossref PubMed Scopus (40) Google Scholar). MARs are believed to connect chromatin loops to the non-chromatin ribonucleoprotein network known as the nuclear matrix (recently reviewed in Ref. 40Nickerson J. J. Cell Sci. 2001; 114: 463-474PubMed Google Scholar). Several reports suggested that sites of DNA synthesis may be linked to the nuclear matrix (41Hozak P. Hassan A.B. Jackson D.A. Cook P.R. Cell. 1993; 73: 361-373Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 42Zink D. Bornfleth H. Visser A. Cremer C. Cremer T. Exp. Cell Res. 1999; 247: 176-188Crossref PubMed Scopus (112) Google Scholar). Thus, TOP1 offers an interesting opportunity to determine whether MARs are binding sites for ORC proteins and whether they function as replication origins. We have also used the ChIP assay to isolate and clone DNA sequences from immunoprecipitated ORC protein-bearing chromatin fragments and identified ORC binding regions in heterochromatic parts of the human genome. Asynchronous HeLa-S3 cells were cultivated on 145-mm dishes in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum. Formaldehyde (Merck) was diluted to 1% in prewarmed medium (37 °C) and added to monolayers of 108cells for 4 min if not otherwise indicated. After removal of the medium, cells were washed 3 times with cold phosphate-buffered saline (PBS), scraped off, washed twice in PBS and resuspended in hypotonic RSB buffer (10 mm Tris, pH 8.0, 3 mmMgCl2, 10 mm sodium bisulfite, pH 8.0) for 10 min on ice. All centrifugation steps were carried out at 600 ×g for 5 min at 4 °C. Cells were disrupted by Dounce homogenization (15 strikes). After centrifugation, nuclear material was washed twice in RSB buffer and once in high salt SNSB buffer (1m NaCl, 10 mm Tris, pH 7.4, 0.1% Nonidet P-40, 1 mm EDTA, 10 mm sodium bisulfite, pH 8.0) and subsequently incubated on ice for 5 min. Finally, the nuclear material was resuspended at physiological salt concentration in NSB buffer (0.1m NaCl, 10 mm Tris, pH 7.4, 0.1% Nonidet P-40, 1 mm EDTA, 10 mm sodium bisulfite, pH 8.0) and loaded onto gradients consisting of 1.3, 1.5, and 1.75 mg/ml CsCl diluted in gradient buffer (0.5% sarcosyl, 1 mm EDTA, 20 mm Tris, pH 8.0). Ultracentrifugation was carried out at 37,000 rpm for 24 h at 18 °C. The nucleoprotein fraction was collected from the gradients followed by overnight dialysis against Tris-EDTA (10 mm Tris, pH 7.4, 1 mm EDTA) supplemented with 10 mm sodium bisulfite, pH 8. Nucleoprotein complexes were sonicated by a total number of 100 short pulses on ice. The concentration of nucleoproteins was determined (A260) and adjusted to 2 μg/μl with Tris-EDTA buffer. Nucleoprotein fragments <1 kb were obtained by treatment with micrococcal nuclease (MBI Fermentas) at 10 units/mg of nucleoprotein in the presence of 3 mm CaCl2for 15 min at 37 °C. The reactions were stopped by adding 20 mm EDTA and analyzed on a 1% agarose gel. Immunoprecipitations were performed with 1 mg of nucleoprotein in NET buffer (50 mm Tris-HCl, pH 7.4, 5 mm EDTA, 150 mm NaCl, 0.5% Nonidet P-40). Affinity-purified antibodies were added at 15 μg (α-ORC1, IgG) and 10 μg (α-ORC2, α-SP1, α-p60/CAF-1) followed by 2-h incubation at 20 °C on a rolling platform. Immunocomplexes were collected by adding 50 μl of 50% protein A-Sepharose and further incubated for 2 h. Coupled protein A-Sepharose beads were washed 8× with radioimmune precipitation buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS), 3× in LiCl2washing buffer (10 mm Tris, pH 8.0, 250 mmLiCl2, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mm EDTA), and 5× in Tris-EDTA buffer. All buffers were supplemented with 10 mm sodium bisulfite, pH 8.0, as protease inhibitor. Beads were transferred to fresh tubes after each buffer change to reduce contamination of unspecific DNA sticking to the tube walls. The washed beads were divided for protein and DNA extraction, respectively. For Western blotting experiments, proteins were eluted with 2% SDS, H2O for 10 min at 37 °C. For a reversal of cross-links, nucleoproteins were incubated for 30 min at 65 °C and extracted with methanol/chloroform (43Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3141) Google Scholar). Input and supernatant samples were treated accordingly. Proteins were separated by SDS page, transferred onto polyvinylidene difluoride membranes, and treated with specific antibodies. Antibodies against human ORC1 and ORC2 have already been described (44Ritzi M. Baack M. Musahl C. Romanowski P. Laskey R.A. Knippers R. J. Biol. Chem. 1998; 273: 24543-24549Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Monospecific antibodies against human ORC3-ORC6 were raised in rabbits using the N-terminal part of the ORC3 protein and the full-length ORC4 protein recombinant expressed in bacteria. Human ORC5 and ORC6 were expressed full-length in insect cells. Antibodies were characterized by immunoblotting using recombinant expressed proteins. To minimize the signal to noise ratio in the PCR, further extensive washing of the coupled protein A-Sepharose beads was crucial. Therefore, the whole washing procedure was repeated as described above. Finally, nucleoproteins were eluted with 1% SDS, Tris-EDTA at 37 °C for 10 min, and proteins were digested with 200 μg/ml proteinase K overnight at 37 °C. DNA was extracted by the standard phenol-chloroform procedure, ethanol-precipitated, and dissolved in 40 μl of Tris-EDTA. Precipitated and extracted DNA was amplified by ligation-mediated-PCR (see Mueller and Wold (45Mueller P.R. Wold B. Science. 1989; 246: 780-786Crossref PubMed Scopus (793) Google Scholar)) using two overlapping linker oligonucleotides (5 pmol/μl/oligonucleotide) 5′-GCGGTGACCCGGGAGATCTGAATTC-3′ and 5′-GAATTCAGATC-3′, which were first annealed to double-stranded DNA by stepwise cooling from 90 °C. For blunt-end ligation, purified DNA fragments were first exposed to the exonuclease activity of the Klenow enzyme (2 units, 5 min, 37 °C). DNA synthesis reactions were started by adding a nucleotide mix (2.5 mmol each of dATP, dCTP, dGTP, dTTP) and further incubated for 30 min. The Klenow enzyme was inactivated at 70 °C for 20 min. DNA fragments were dephosphorylated with 1 unit of alkaline phosphatase at 37 °C for 1 h. Linker ligations were performed at 18 °C for 15 h using 1 unit of T4-DNA ligase and 2 μl of the double-stranded linker oligonucleotides. PCR reactions were performed in the presence of 10 mm each dideoxynucleotide, dATP, dCTP, dGTP, dTTP, 3 units of Pfu DNA polymerase, 6% glycerol, and 25 pmol of each oligonucleotide. The PCR was performed in a thermocycler at 30 cycles consisting of 1 min at 94 °C, 2 min at 63 °C, and 3 min at 72 °C. Amplified DNA fragments were directly cloned in the pCR-BluntII-TOPO cloning system (Invitrogen) according to the manufacturer's manual. Plasmid DNA was extracted from bacteria, purified, and analyzed by sequencing and PCR methods. Approximately 1 × 108 HeLa S3 cells were trypsinized and washed twice in ice-cold phosphate-buffered saline and once in RBS (10 mmTris, pH 7.4, 10 mm NaCl, 3 mmMgCl2). Centrifugations were carried out at 600 ×g for 10 min. Cells were resuspended in RBS on ice at about 5 × 106 cells/ml for 5 min. The same volume of 1% Nonidet P-40, RBS was added, and cells were further incubated on ice for 10 min. The nuclei were pelleted, washed twice in RBS, and resuspended at 5 × 107 nuclei/ml. The same volume of lysis buffer (20 mm Tris, pH 8.0, 20 mm EDTA, 2% SDS, 500 μg/ml proteinase K) was added and incubated overnight at 56 °C. Total genomic DNA was extracted with phenol/chloroform, precipitated with isopropanol, and dissolved in Tris-EDTA buffer at 2 μg/μl. DNA was denatured at 85 °C for 10 min followed by rapid cooling on ice and loaded on 5–30% (w/v) linear neutral sucrose gradients in Tris-EDTA buffer (plus 0.1 m NaCl). In a parallel tube, double-stranded size marker DNA (1-kb latter, MBI Fermentas) was loaded as a reference. Gradients were centrifuged at 20 °C in a Beckman SW28 rotor for 20 h at 26,000 rpm. Fractions of 1 ml were collected from top to bottom. The distribution of size markers in the gradient fractions was determined by agarose gel electrophoresis. DNA fractions corresponding to an average of 1-kb size (nascent DNA strands) and 2–10 kb were collected and precipitated with ethanol. The abundance of nascent DNA strands in the preparation was determined by quantitative real-time PCR. Real-time PCR was performed with the Light Cycler instrument (Roche Molecular Biochemicals) using a ready-to-use “hot start” reaction mix. The mix containsTaq DNA polymerase and a fluorescent dye, SYBR Green I, for real-time detection of double-stranded DNA. Reactions were set up in 10 μl including 0.5 mm each primer. PCR reactions were performed at 50 cycles routinely, using the standard settings recommended by Roche Molecular Biochemicals. Annealing temperatures of individual primers are indicated in Table I. Standard DNA samples (human genomic DNA) were serially diluted to 30 ng, 3 ng, 300 pg, 30 pg, and 3 pg. After PCR, the x axis crossing point of each standard sample was plotted against the logarithm of concentration to produce a standard curve. Genomic equivalents of DNA samples were determined by extrapolation from the standard curve (36Ladenburger E.-M. Keller C. Knippers R. Mol. Cell. Biol. 2002; 22: 1036-1048Crossref PubMed Scopus (130) Google Scholar).Table IOligonucleotides used for quantitative real-time PCR assays Covalent cross-linking of chromatin proteins to DNAin vivo, and the isolation of cross-linked chromatin was performed as described (46Crane-Robinson C. Myers F.A. Hebbes T.R. Clayton A.L. Thorne A.W. Methods Enzymol. 1999; 304: 533-547Crossref PubMed Scopus (40) Google Scholar, 47Orlando V. Trends Biochem. Sci. 2000; 25: 99-104Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 48Göhring F. Fackelmayer F.O. Biochemistry. 1997; 36: 8276-8283Crossref PubMed Scopus (81) Google Scholar). Isolated chromatin was sonicated and further trimmed by micrococcal nuclease to produce fragments with DNA of 0.2–1-kbp lengths (Fig.1A). Chromatin fragments were either directly prepared for polyacrylamide gel electrophoresis and immunoblotting (input, Fig. 1) or first immunoprecipitated with specific antibodies (precipitate, Fig. 1) and then analyzed by immunoblotting. Immunoblots showed that a cross-linking time of 4 min was sufficient to covalently link hOrc2p to DNA (44Ritzi M. Baack M. Musahl C. Romanowski P. Laskey R.A. Knippers R. J. Biol. Chem. 1998; 273: 24543-24549Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). This was also an optimal cross-linking time for other proteins such as the chromatin-associated fraction of the single strand-specific DNA-binding protein RPA and the scaffold attachment factor scaffold attachment factor A (SAF-A), an abundant nuclear protein that is bound to MAR elements in vivo (Ref. 49Fackelmayer F.O. Richter A. Biochemistry. 1994; 33: 10416-10422Crossref PubMed Scopus (49) Google Scholar; Fig. 1B, input). Orc2-specific antibodies efficiently precipitated chromatin fragments with covalently bound hOrc2p, but these precipitates contained little if any RPA and no detectable SAF-A (Fig. 1B, precipitate), indicating that hOrc2p and SAF-A were not cross-linked to the same chromatin fragments and, therefore, do most probably not reside at closely adjacent chromatin sites in vivo. Fig. 1C shows the cross-linking to DNA of other nuclear proteins such as the p60 subunit of the chromatin assembly factor CAF1 (50Smith S. Stillman B. Cell. 1989; 58: 15-25Abstract Full Text PDF PubMed Scopus (515) Google Scholar) and transcription factor Sp1 (51Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1248) Google Scholar). Immunoprecipitations with Orc2-specific antibodies indicate that the p60 subunit was not present in these immunoprecipitates (Fig. 1C). Interestingly, a fraction of transcription factor Sp1 always co-precipitated with hOrc2p-bearing chromatin (Fig. 1C), probably indicating that Sp1 and hOrc2p were occasionally cross-linked to the same chromatin fragments (see below). Vashee et al. (53Vashee S. Simancek P. Challberg M.D. Kelly T.J. J. Biol. Chem. 2001; 276: 26666-26673Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and Dhar et al. (52Dhar S.K. Delmolino L. Dutta A. J. Biol. Chem. 2001; 276: 29067-29071Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) show that human ORCs including subunits Orc1p-Orc5p can be extracted at high salt from HeLa cell chromatin and that human proteins Orc2p-Orc5p form a core complex to which proteins hOrc1p and hOrc6p are more loosely bound. It was, therefore, of interest to determine whether the other ORC proteins could be immunoprecipitated together with hOrc2p by Orc2-specific antibodies. We first determined whether the six ORC proteins in asynchronously proliferating human cells could be identified by the available antibodies. For that purpose, HeLa cells were fractionated to yield cytosol (Cy), soluble nuclear proteins (Nu), and chromatin, which was treated with increasing salt (Fig.2A). The six ORC proteins were detected on chromatin and could be mobilized with 0.1–0.25m NaCl. However, a fraction of hOrc1p appeared to be more stably bound to chromatin since higher salt concentrations were required for an efficient elution. We also detected significant amounts of hOrc6p in soluble protein fractions (Fig. 2A). Next we investigated whether the ORC subunits could be cross-linked to DNA. On isolated chromatin we detected significant amounts of subunits hOrc1p (Fig. 1) as well as hOrc2p–hOrc5p but reduced amounts of hOrc6p (Fig. 2B, input), which is in agreement with published data, suggesting that hOrc6p may not be a regular component of human ORC (52Dhar S.K. Delmolino L. Dutta A. J. Biol. Chem. 2001; 276: 29067-29071Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 53Vashee S. Simancek P. Challberg M.D. Kelly T.J. J. Biol. Chem. 2001; 276: 26666-26673Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Cross-linked chromatin was then immunoprecipitated with Orc2p-specific antibodies. The precipitates clearly contained hOrc1 as well as hOrc2p–hOrc4p (Fig. 2B) but very little, if any, hOrc6p (Fig. 2B). The Orc5p band overlapped to a significant extent with the IgG heavy chain band in the experiment. However, in independent experiment we clearly detected an Orc5p band in the immunoprecipitate. Comparing the input and supernatant, we estimate that between 30 and 50% of the cross-linked input sample could be immunoprecipitated. No ORC proteins were precipitated with unspecific IgGs (Fig. 2B) or with p60-specific antibodies (not shown). We have also tested antibodies against Orc3p, Orc4p, and Orc5p and found that the antibodies did not efficiently precipitate cross-linked proteins (not shown). Therefore, we used the Orc1p- and Orc2p-specific antibodies for the experiments reported below. Thus, ORC proteins could be covalently linked to chromatin and most likely occurred at the same chromatin sites either as one large complex or, alternatively, as subcomplexes. Next we addressed the question of whether ORC binds to particular genomic regions. The well characterized TOP1 gene locus appeared to be a interesting region because it contains several features that have frequently been found in known origins such as A+T-rich elements, MARs, nuclease hypersensitive sites, and a higher than average G+C content in the gene promoter. The TOP1 gene is composed of 21 exons (37Kunze N. Yang G.C. Dolberg M. Sundarp R. Knippers R. Richter A. J. Biol. Chem. 1991; 266: 9610-9616Abstract Full Text PDF PubMed Google Scholar) and contains MAR I, located at an intronic site immediately after exon 2, and MAR II, which occurs further downstream, between exons 13 and 14 (Ref. 39Romig H. Ruff J. Fackelmayer F.O. Patil M.S. Richter A. Eur. J. Biochem. 1994; 221: 411-419Crossref PubMed Scopus (40) Google Scholar; Fig.3). MAR I and MAR II specifically attach to components of the nuclear matrix and exhibit specific binding sites for the SAF-A both in vitro and in vivo (39Romig H. Ruff J. Fackelmayer F.O. Patil M.S. Richter A. Eur. J. Biochem. 1994; 221: 411-419Crossref PubMed Scopus (40) Google Scholar,49Fackelmayer F.O. Richter A. Biochemistry. 1994; 33: 10416-10422Crossref PubMed Scopus (49) Google Scholar). The TOP1 promoter is composed of several elements that function as binding sites for transcription factors including the ubiquitous Sp1 protein (54Heiland S. Knippers R. Mol. Cell. Biol. 1995; 15: 6623-6631Crossref PubMed Google Scholar). The promoter has a G+C content of 67% and co-localizes with a CpG island. This is of interest because it is assumed that origins of DNA replications are predominantly located in the vicinity of CpG islands (55Antequera F. Bird A. Curr. Biol. 1999; 9: 661-667Abstract Full Text Full Text PDF PubMed Scopus (182) Google
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