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• W2045607467 abstract "Understanding protein-DNA interactions in vivo at origins of DNA replication throughout the cell cycle may shed further insight on the mechanisms of initiation and replication control. The Burkitt's lymphoma cell line Raji harbors multiple copies of latent Epstein-Barr virus. Once per cell cycle the origin of plasmid replication of Epstein-Barr virus provides replication function in cis for the viral DNA. Here we examined in vivo nucleoprotein complexes on the initiator element of the origin before and after DNA synthesis. For this purpose Raji cells were synchronously growth arrested in G1 phase by mimosine and in mitosis by colchicine, respectively. The association of the initiator element with proteins was visualized by footprinting with dimethyl sulfate and ligation mediated polymerase chain reaction. Methylation patterns indicated a novel binding activity within each element of a nonamer repeated three times at the initiator element. This activity was strongly diminished in mitotic cells. Furthermore, 5′-ends of Epstein-Barr virus DNA were mapped to the nonamers by ligation mediated polymerase chain reaction, suggesting potential initiation sites for replication from DS. Understanding protein-DNA interactions in vivo at origins of DNA replication throughout the cell cycle may shed further insight on the mechanisms of initiation and replication control. The Burkitt's lymphoma cell line Raji harbors multiple copies of latent Epstein-Barr virus. Once per cell cycle the origin of plasmid replication of Epstein-Barr virus provides replication function in cis for the viral DNA. Here we examined in vivo nucleoprotein complexes on the initiator element of the origin before and after DNA synthesis. For this purpose Raji cells were synchronously growth arrested in G1 phase by mimosine and in mitosis by colchicine, respectively. The association of the initiator element with proteins was visualized by footprinting with dimethyl sulfate and ligation mediated polymerase chain reaction. Methylation patterns indicated a novel binding activity within each element of a nonamer repeated three times at the initiator element. This activity was strongly diminished in mitotic cells. Furthermore, 5′-ends of Epstein-Barr virus DNA were mapped to the nonamers by ligation mediated polymerase chain reaction, suggesting potential initiation sites for replication from DS. INTRODUCTIONPlasmid replication of Epstein-Barr virus (EBV)1( 1The abbreviations used are: EBVEpstein-Barr virusoriPorigin of plasmid replicationS phasesynthesis phaseEBNA1Epstein-Barr viral nuclear antigen 1FRfamily of repeatsDSdyad symmetryLMPCRligation mediated polymerase chain reactionPBSphosphate-buffered salineFACScanfluorescence-activated cell scanDMSdimethyl sulfatedNTPdeoxynucleotide triphosphateMmitosis.) may serve as a viral model system for chromosomal DNA replication in higher eukaryotes (DePamphilis, 1988DePamphilis M.L. Cell. 1988; 52: 635-638Abstract Full Text PDF PubMed Scopus (201) Google Scholar). EBV persists in lymphoid cells, such as the Raji cell line derived from an African Burkitt's lymphoma, in a tightly latent state as a circular plasmid in multiple copies (Adams and Lindahl, 1975Adams A. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1477-1481Crossref PubMed Scopus (100) Google Scholar; Lindahl et al., 1976Lindahl T. Adams A. Bjursell G. Bornkamm G.W. Kaschka-Dierich C. Jehn U. J. Mol. Biol. 1976; 102: 511-530Crossref PubMed Scopus (199) Google Scholar; Nonoyama and Pagano, 1972Nonoyama M. Pagano J.S. Nat. New Biol. 1972; 238: 169-171Crossref PubMed Scopus (111) Google Scholar). Like cellular chromosomes, each viral plasmid is replicated once per cell cycle in the early synthesis (S) phase (Adams, 1987Adams A. J. Virol. 1987; 61: 1743-1746Crossref PubMed Google Scholar; Gussander and Adams, 1984Gussander E. Adams A. J. Virol. 1984; 52: 549-556Crossref PubMed Google Scholar; Hampar et al., 1974Hampar B. Tanaka A. Nonoyama M. Derge J.G. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 631-635Crossref PubMed Scopus (53) Google Scholar; Yates and Guan, 1991Yates J.L. Guan N. J. Virol. 1991; 65: 483-488Crossref PubMed Google Scholar). The replication of EBV-derived plasmids is dependent on oriP, the viral protein EBNA1, and on a set of mostly unknown cellular proteins (Lupton and Levine, 1985Lupton S. Levine A.J. Mol. Cell. Biol. 1985; 5: 2533-2542Crossref PubMed Scopus (209) Google Scholar; Reisman et al., 1985Reisman D. Yates J. Sugden B. Mol. Cell. Biol. 1985; 5: 1822-1832Crossref PubMed Scopus (312) Google Scholar; Yates et al., 1984Yates J. Warren N. Reisman D. Sugden B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3806-3810Crossref PubMed Scopus (487) Google Scholar, Yates et al., 1985Yates J.L. Warren N. Sugden B. Nature. 1985; 313: 812-815Crossref PubMed Scopus (979) Google Scholar). OriP consists of two sequence elements, the family of repeat element (FR) and the dyad symmetry element (DS), separated by a stretch of about 960 base pairs of unique DNA dispensable for ori function (Lupton and Levine, 1985Lupton S. Levine A.J. Mol. Cell. Biol. 1985; 5: 2533-2542Crossref PubMed Scopus (209) Google Scholar; Reisman et al., 1985Reisman D. Yates J. Sugden B. Mol. Cell. Biol. 1985; 5: 1822-1832Crossref PubMed Scopus (312) Google Scholar). FR contains 20 times a 30-base pair repeat in tandem (Lupton and Levine, 1985Lupton S. Levine A.J. Mol. Cell. Biol. 1985; 5: 2533-2542Crossref PubMed Scopus (209) Google Scholar; Reisman et al., 1985Reisman D. Yates J. Sugden B. Mol. Cell. Biol. 1985; 5: 1822-1832Crossref PubMed Scopus (312) Google Scholar), each containing an EBNA1 binding site (Hsieh et al., 1993Hsieh D.-J. Camiolo S.M. Yates J.L. EMBO J. 1993; 12: 4933-4944Crossref PubMed Scopus (77) Google Scholar; Rawlins et al., 1985Rawlins D.R. Milman G. Hayward S.D. Hayward G.S. Cell. 1985; 42: 859-868Abstract Full Text PDF PubMed Scopus (341) Google Scholar). In EBNA1 expressing primate cells, FR is essential for the stable maintenance of EBV plasmids (Lupton and Levine, 1985Lupton S. Levine A.J. Mol. Cell. Biol. 1985; 5: 2533-2542Crossref PubMed Scopus (209) Google Scholar; Reisman et al., 1985Reisman D. Yates J. Sugden B. Mol. Cell. Biol. 1985; 5: 1822-1832Crossref PubMed Scopus (312) Google Scholar; Wysokenski and Yates, 1989Wysokenski D.A. Yates J.L. J. Virol. 1989; 63: 2657-2666Crossref PubMed Google Scholar). The element contains the termination site for replication (Dhar and Schildkraut, 1991Dhar V. Schildkraut C.L. Mol. Cell. Biol. 1991; 11: 6268-6278Crossref PubMed Scopus (42) Google Scholar; Gahn and Schildkraut, 1989Gahn T.A. Schildkraut C.L. Cell. 1989; 58: 527-535Abstract Full Text PDF PubMed Scopus (226) Google Scholar) and works as a replication enhancer (Wysokenski and Yates, 1989Wysokenski D.A. Yates J.L. J. Virol. 1989; 63: 2657-2666Crossref PubMed Google Scholar). Furthermore, it functions as a transcriptional enhancer (Reisman and Sugden, 1986Reisman D. Sugden B. Mol. Cell. Biol. 1986; 6: 3838-3846Crossref PubMed Scopus (252) Google Scholar; Wysokenski and Yates, 1989Wysokenski D.A. Yates J.L. J. Virol. 1989; 63: 2657-2666Crossref PubMed Google Scholar). DS contains four EBNA1 binding sites and a region of dyad symmetry encompassing EBNA1 binding sites four and three (Hsieh et al., 1993Hsieh D.-J. Camiolo S.M. Yates J.L. EMBO J. 1993; 12: 4933-4944Crossref PubMed Scopus (77) Google Scholar; Lupton and Levine, 1985Lupton S. Levine A.J. Mol. Cell. Biol. 1985; 5: 2533-2542Crossref PubMed Scopus (209) Google Scholar; Rawlins et al., 1985Rawlins D.R. Milman G. Hayward S.D. Hayward G.S. Cell. 1985; 42: 859-868Abstract Full Text PDF PubMed Scopus (341) Google Scholar; Reisman et al., 1985Reisman D. Yates J. Sugden B. Mol. Cell. Biol. 1985; 5: 1822-1832Crossref PubMed Scopus (312) Google Scholar). In EBNA1 expressing primate cells DS most likely functions as the physical origin of bidirectional replication of EBV-derived plasmids (Gahn and Schildkraut, 1989Gahn T.A. Schildkraut C.L. Cell. 1989; 58: 527-535Abstract Full Text PDF PubMed Scopus (226) Google Scholar; Platt et al., 1993Platt T.H.K. Tcherepanova I.Y. Schildkraut C.L. J. Virol. 1993; 67: 1739-1745Crossref PubMed Google Scholar; Wysokenski and Yates, 1989Wysokenski D.A. Yates J.L. J. Virol. 1989; 63: 2657-2666Crossref PubMed Google Scholar). Since initiation of DNA replication from oriP is a strictly regulated and dynamic event, we asked if there are variations in the proteins associated with the initiator element of oriP in vivo before and after S phase. Therefore, we decided to examine the state of in vivo protein-DNA interactions at the initiator element of oriP in synchronously before and after S phase growth-arrested Raji cells. Methylation protected and sensitive nucleotides were visualized using the technique of ligation mediated polymerase chain reaction (LMPCR) (Mueller and Wold, 1989Mueller P.R. Wold B. Science. 1989; 246: 780-786Crossref PubMed Scopus (792) Google Scholar). The exact physical initiation points or areas of EBV plasmid replication are not yet known at nucleotide resolution. Therefore, we decided to use the same method to visualize 5′-ends of viral DNA within the initiator element of oriP.EXPERIMENTAL PROCEDURESTissue CultureRaji cells were maintained in suspension cultures of RPMI 1640 medium containing 10% fetal calf serum, 2 mM glutamine, 50 units of penicillin/ml, and 50 μg of streptomycin/ml under 5% CO2 and 37°C.Flow Cytometric AnalysisFor flow cytometric analysis cells were harvested, fixed with 70% methanol for at least 1 h, resuspended in 1 ml of phosphate-buffered saline (PBS) (Saluz and Jost, 1990Saluz H.P. Jost J.P. A Laboratory Guide for in Vivo Studies of DNA Methylation and Protein/DNA Interactions. Birkhäuser, Basel1990Crossref Google Scholar), digested with 100 μg of RNase A for 1 h at 37°C, stained with 50 μl of propidium iodide (1 mg/ml), and scanned on a Becton Dickinson FACScan analyzer using Cellfit software.DMS in Vivo FootprintingFor each footprint 107 cells were harvested by centrifugation, washed with PBS (Saluz and Jost, 1990Saluz H.P. Jost J.P. A Laboratory Guide for in Vivo Studies of DNA Methylation and Protein/DNA Interactions. Birkhäuser, Basel1990Crossref Google Scholar), resuspended in 1 ml of PBS, and incubated at room temperature for 5 min with 10 μl of dimethyl sulfate (DMS). The reaction was stopped by the addition of 5 ml of ice-cold DMS stop solution (1% of bovine serum albumin and 100 μM β-mercaptoethanol dissolved in PBS) (Saluz and Jost, 1990Saluz H.P. Jost J.P. A Laboratory Guide for in Vivo Studies of DNA Methylation and Protein/DNA Interactions. Birkhäuser, Basel1990Crossref Google Scholar). Cells were pelleted and washed once more with DMS stop solution and two more times with PBS. Cells were resuspended in 1 ml of PBS. Genomic DNAs were isolated according to standard methods (Saluz and Jost, 1990Saluz H.P. Jost J.P. A Laboratory Guide for in Vivo Studies of DNA Methylation and Protein/DNA Interactions. Birkhäuser, Basel1990Crossref Google Scholar; Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), either before sequencing reactions or after DMS treatment of cells. DMS-treated purified DNA was subjected to piperidine treatment (Maxam and Gilbert, 1980Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 497-560Crossref PubMed Scopus (4) Google Scholar). For visualization by LMPCR, 2 μg of sequenced or footprinted DNA were analyzed according to the protocol of Garrity and Wold, 1992Garrity P.A. Wold B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1021-1025Crossref PubMed Scopus (202) Google Scholar with some modifications. The following sets of oligonucleotides i, ii, and iii for the LMPCR assay of both strands were used, 5′ to 3′: lower strand (i) 8927-GGTTCACTACCCTCGTGGAATCCTG-8951, (ii) 8931-CACTACCCTCGTGGAATCCTGACCC-8955, (iii) 8969-CCGTGACAGCTCATGGGGTGGGAGATATC-8997; upper strand (i) 9229-GGCTACACCAACGTCAATCAGAGGG-9205, (ii) 9205-GGCCTGTGTAGCTACCGATAAGCGG-9181, (iii) 9195-GCTACCGATAAGCGGACCCTCAAGAGG-9169. The first strand primer extension reaction was done in 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8, 25°C (Vent buffer, New England Biolabs), containing 0.3 pmol of primer i, 240 μM each dNTP, and 1 unit of Vent (exo-) DNA polymerase (New England Biolabs) for 5 min at 94°C, 30 min at 60°C, and 10 min at 72°C. For ligation of the common linker, the sample was transferred to ice and 5 μl of PCR linker mixture as in Mueller and Wold, 1989Mueller P.R. Wold B. Science. 1989; 246: 780-786Crossref PubMed Scopus (792) Google Scholar, 2 μl of ligation buffer (660 mM Tris-HCl, 50 mM MgCl2, 10 mM dithioerythritol, 10 mM ATP, pH 7.5, 20°C, Boehringer Mannheim), 1 μl of T4 DNA-ligase (5 units/μl, Boehringer Mannheim), and 12 μl of water were added. After an overnight incubation at 4°C the DNA was ethanol precipitated, washed once with 75% ethanol, dried, and then resuspended in water. The PCR amplification was done in 100 μl of Vent buffer containing 10 pmol each of primer ii and the longer linker primer, 240 μM each dNTP, and 3 units of Vent (exo-) DNA polymerase for 20 cycles using 1 min at 94°C, 1.5 min at 60°C, and 3 min at 72°C. For labeling, the sample was transferred to ice, 5 pmol of T4 kinase [γ-32P]ATP labeled primer iii, 2.5 nmol each dNTP, and 0.5 units of Vent (exo-) DNA polymerase in a volume of Vent buffer not exceeding 15 μl were added. Then the sample was heated to 94°C for 1.5 min, subjected to 5 cycles of 2 min at 94°C, 2 min at 62°C, and 5 min at 72°C, and kept at 72°C for 5 more minutes. Samples were transferred to ice, phenol/chloroform extracted, ethanol precipitated, and resuspended in loading dye. One-fifth of each sample was separated on a standard 6% sequencing gel (Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), and the gels were dried and autoradiographed for 15 h at room temperature with Kodak X-Omat LS film.Visualization of 5′-EndsGenomic DNA was harvested from unsynchronized Raji cells, and from Raji cells treated with 30 μM aphidicolin for up to 24 h (Saluz and Jost, 1990Saluz H.P. Jost J.P. A Laboratory Guide for in Vivo Studies of DNA Methylation and Protein/DNA Interactions. Birkhäuser, Basel1990Crossref Google Scholar). 5′-Ends of DNA were visualized by LMPCR on 2 μg of genomic DNA using the same reaction conditions and primer sets as for the in vivo footprints.RESULTSSynchronous Growth Arrest of Raji CellsInitial efforts were focussed on the synchronization of Raji cells. The cells used in our experiments were growing fast, having a doubling time of about 24 h under standard cell culture conditions. For the purpose of arresting cells before and after S phase, we used the drugs mimosine and colchicine, respectively. Colchicine arrests mitotic cells in the metaphase by blocking the spindle apparatus for the separation of the chromosomes (Inoué, 1981Inoué S. J. Cell Biol. 1981; 91: 131s-147sCrossref PubMed Google Scholar). Mimosine, a plant amino acid, has the potential to reversibly block the mammalian cell cycle close to the G1/S boundary (Lalande, 1990Lalande M. Exp. Cell Res. 1990; 186: 332-339Crossref PubMed Scopus (178) Google Scholar). The cell cycle block by mimosine may be due to the inhibition of initiation of DNA replication (Mosca et al., 1992Mosca P.J. Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1992; 12: 4375-4383Crossref PubMed Scopus (123) Google Scholar). Drugs were added to exponentially growing cells. The flow cytometric analysis of untreated, propidium iodide-stained cells showed a relatively high DNA synthesis rate and a relatively large fraction of the cells in G2/M (Fig. 1A). 14 h later, when the cells were harvested for the footprints, a control sample analyzed by flow cytometry showed essentially the same pattern (Fig. 1b). Cells treated with 400 μM mimosine or with 1 μM colchicine for 14 h, respectively, showed a rather different distribution over the cell cycle (Fig. 1, C and D). In the mimosine-treated sample, S phase cells and G2/M cells were strongly depleted and a large majority of the cells were in G1 (Fig. 1C). In the colchicine-treated sample, G1 cells and S phase cells were strongly depleted and the vast majority of the cells were in G2/M (Fig. 1D). These two cell cycle arrests seemed sufficiently quantitative for our intended studies of protein-DNA interactions at DS of oriP before and after S phase.EBNA1 Binding to DS Throughout the Cell CycleCells treated with mimosine and colchicine were harvested and genomic DMS footprints on the initiator element (DS) of oriP of EBV were performed (Fig. 2). The footprinting was done on both strands of DS according to standard methods (Garrity and Wold, 1992Garrity P.A. Wold B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1021-1025Crossref PubMed Scopus (202) Google Scholar; Maxam and Gilbert, 1980Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 497-560Crossref PubMed Scopus (4) Google Scholar; Mueller and Wold, 1989Mueller P.R. Wold B. Science. 1989; 246: 780-786Crossref PubMed Scopus (792) Google Scholar; Saluz and Jost, 1990Saluz H.P. Jost J.P. A Laboratory Guide for in Vivo Studies of DNA Methylation and Protein/DNA Interactions. Birkhäuser, Basel1990Crossref Google Scholar; Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) with some modifications. The pattern of guanines protected from methylation and nucleotides hypersensitive to methylation is summarized in Fig. 4. The footprint patterns for mimosine-treated cells and colchicine-treated cells were similar, in general, and consistent with the concept of EBNA1 binding to its four in vitro binding sites (Rawlins et al., 1985Rawlins D.R. Milman G. Hayward S.D. Hayward G.S. Cell. 1985; 42: 859-868Abstract Full Text PDF PubMed Scopus (341) Google Scholar) within DS also in vivo (Hsieh et al., 1993Hsieh D.-J. Camiolo S.M. Yates J.L. EMBO J. 1993; 12: 4933-4944Crossref PubMed Scopus (77) Google Scholar). Nuclear proteins other than EBNA1 might also cause the in vivo footprints on the EBNA1 binding sites at some phase of the cell cycle (Oh et al., 1991Oh S.-J. Chittenden T. Levine A.J. J. Virol. 1991; 65: 514-519Crossref PubMed Google Scholar; Wen et al., 1990Wen L.-T. Lai P.K. Bradley G. Tanaka A. Nonoyama M. Virology. 1990; 178: 293-296Crossref PubMed Scopus (7) Google Scholar). However, since the footprints spanning the EBNA1 binding sites of DS were the same in mimosine- and colchicine-treated cells and also in nontreated, exponentially growing cells (data not shown), we concluded that EBNA1 binding to DS is unchanged throughout the cell cycle (Hsieh et al., 1993Hsieh D.-J. Camiolo S.M. Yates J.L. EMBO J. 1993; 12: 4933-4944Crossref PubMed Scopus (77) Google Scholar). Since EBNA1 binds on only one face of the double helix within DS (Frappier and O'Donnell, 1992Frappier L. O'Donnell M. J. Virol. 1992; 66: 1786-1790Crossref PubMed Google Scholar; Kimball et al., 1989Kimball A.S. Milman G. Tullius T.D. Mol. Cell. Biol. 1989; 9: 2738-2742Crossref PubMed Scopus (12) Google Scholar), the protections of guanines 9037, 9044, 9065, and 9119 are not readily explained solely by the binding of EBNA1. These protections may be caused by structural alterations of the DNA induced by the binding of EBNA1 (Frappier and O'Donnell, 1992Frappier L. O'Donnell M. J. Virol. 1992; 66: 1786-1790Crossref PubMed Google Scholar), or also by the presence of nuclear proteins other than EBNA1 in the nucleoprotein complexes on DS (Oh et al., 1991Oh S.-J. Chittenden T. Levine A.J. J. Virol. 1991; 65: 514-519Crossref PubMed Google Scholar; Wen et al., 1990Wen L.-T. Lai P.K. Bradley G. Tanaka A. Nonoyama M. Virology. 1990; 178: 293-296Crossref PubMed Scopus (7) Google Scholar).Figure 2:Genomic footprinting on both strands of the dyad symmetry element of oriP with dimethyl sulfate. Panel a shows data for the lower strand, panel b for the upper strand. Lanes A+G and G refer to Maxam and Gilbert, 1980Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 497-560Crossref PubMed Scopus (4) Google Scholar sequencing reactions using purified genomic DNA. Lanes Co and Mi are DMS footprints done on live cells treated with colchicine and mimosine, respectively, as shown in Fig. 1. Experiments were done several times from independent synchronous growth arrests with the same results. The numbers on the left of each panel refer to the EBV sequence (Baer et al., 1984Baer R. Bankier A.T. Biggin M.D. Deininger P.L. Farrell P.J. Gibson T.J. Hatfull G. Hudson G.S. Satchwell S.C. Sequin C. Tuffnell P.S. Barrell B.G. Nature. 1984; 310: 207-211Crossref PubMed Scopus (1444) Google Scholar), the numbers on the right of each panel refer to the EBNA1 binding sites within DS (Rawlins et al., 1985Rawlins D.R. Milman G. Hayward S.D. Hayward G.S. Cell. 1985; 42: 859-868Abstract Full Text PDF PubMed Scopus (341) Google Scholar), nonamers are indicated by the letters A, B, and C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4:Summary of genomic footprinting (Fig. 2) and free 5′-ends (Fig. 3) on the dyad symmetry element of oriP of Epstein-Barr virus in Raji cells. The positions of protections and enhanced cleavage sites as presented in Fig. 2 are shown here for G1 and G2/M cells. EBNA1 binding sites (Ambinder et al., 1990Ambinder R.F. Shah W.A. Rawlins D.R. Hayward G.S. Hayward S.D. J. Virol. 1990; 64: 2369-2379Crossref PubMed Google Scholar) and nonamers (see text) are indicated by horizontal lines drawn between the DNA strands. Numbering of the sequence and the EBNA1 sites refers to Baer et al., 1984Baer R. Bankier A.T. Biggin M.D. Deininger P.L. Farrell P.J. Gibson T.J. Hatfull G. Hudson G.S. Satchwell S.C. Sequin C. Tuffnell P.S. Barrell B.G. Nature. 1984; 310: 207-211Crossref PubMed Scopus (1444) Google Scholar and Rawlins et al., 1985Rawlins D.R. Milman G. Hayward S.D. Hayward G.S. Cell. 1985; 42: 859-868Abstract Full Text PDF PubMed Scopus (341) Google Scholar, respectively, the dyad symmetry (Lupton and Levine, 1985Lupton S. Levine A.J. Mol. Cell. Biol. 1985; 5: 2533-2542Crossref PubMed Scopus (209) Google Scholar; Reisman et al., 1985Reisman D. Yates J. Sugden B. Mol. Cell. Biol. 1985; 5: 1822-1832Crossref PubMed Scopus (312) Google Scholar) is indicated by horizontal arrows, nonamers are labeled A, B, and C. Differences between this genomic sequence and the published B95-8 sequence (Baer et al., 1984Baer R. Bankier A.T. Biggin M.D. Deininger P.L. Farrell P.J. Gibson T.J. Hatfull G. Hudson G.S. Satchwell S.C. Sequin C. Tuffnell P.S. Barrell B.G. Nature. 1984; 310: 207-211Crossref PubMed Scopus (1444) Google Scholar) are indicated by small letters above and below the respective DNA strand. Guanines strongly protected from methylation by DMS are indicated by filled circles, weakly protected guanines are indicated by open circles, enhanced reactivity to DMS is shown by filled and open triangles, analogously. The pattern of DMS reactivity for colchicine-treated cells is shown for the entire sequence, the different reactivities of the nonamer in mimosine-treated cells for the upper and lower strand, respectively, are indicated above and below the symbols for reactivities of colchicine-treated cells. Free 5′-ends at nonamers in genomic DNA from untreated cells visualized by LMPCR are indicated by small vertical arrows above and below the respective strand.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Novel Protein Binding at DSWe observed differences between previously published DMS footprints on DS from nonsynchronized cells (Hsieh et al., 1993Hsieh D.-J. Camiolo S.M. Yates J.L. EMBO J. 1993; 12: 4933-4944Crossref PubMed Scopus (77) Google Scholar) and this data. First, there was a guanine instead of an adenine at position 9062 in oriP of our Raji cells, that was methylation protected. This transitional point mutation is unlikely to have any effects on the function of oriP (Ambinder et al., 1990Ambinder R.F. Shah W.A. Rawlins D.R. Hayward G.S. Hayward S.D. J. Virol. 1990; 64: 2369-2379Crossref PubMed Google Scholar). Second, the adenine at position 9051 was strongly DMS reactive. This difference may be due to variations in technical procedures. The methylation sensitive adenine at 9051 has also been observed in in vitro methylation studies on the distortion of oriP by EBNA1 (Frappier and O'Donnell, 1992Frappier L. O'Donnell M. J. Virol. 1992; 66: 1786-1790Crossref PubMed Google Scholar). Third, and more important, we observed a novel pattern of DMS reactivity within DS that was not reported by the earlier paper (Hsieh et al., 1993Hsieh D.-J. Camiolo S.M. Yates J.L. EMBO J. 1993; 12: 4933-4944Crossref PubMed Scopus (77) Google Scholar). This discrepancy may be explained by variations in technical procedures or also by differences between Raji cells that have been passaged for many years.Cell Cycle DependenceThe novel pattern was observed in nonsynchronized cells (data not shown) in a way similar to mimosine-treated cells. However, the pattern was different between mimosine- and colchicine-treated cells. The pattern was found at each nonamer, 5′-TTAGGGTTA-3′, repeated three times within DS. The nonamers are located at 9021 to 9029 (A), 9073 to 9081 (B) and 9127 to 9135 (C), each location being outside of the EBNA1 sites. Nonamer A points to one direction, nonamers B and C point to the other direction. The first guanine of each nonamer was protected stronger in mimosine than in colchicine-treated cells. The third guanine each was slightly stronger methylation sensitive in mimosine- than in colchicine-treated cells. These differences in the footprint patterns between mimosine- and colchicine-treated cells most likely reflect the differential interaction of the nonamer with nuclear protein or, alternatively, a more general change of the nucleoprotein complex on DS. An earlier experimental hint for protein binding to the nonamer sequence came from in vitro footprints on DS. Nuclear proteins from HeLa cells yielded distinct DNase I protected areas within DS that spanned the nonamers (Oh et al., 1991Oh S.-J. Chittenden T. Levine A.J. J. Virol. 1991; 65: 514-519Crossref PubMed Google Scholar). Therefore, distinct nucleoprotein complexes on DS probably exist in Raji cells in vivo.DNA 5′-Ends at the NonamersIn order to correlate more the protein binding data at DS with initiation function, we tried to locate the initiation points of DNA replication within DS at nucleotide resolution. Therefore, we performed LMPCR on purified genomic DNA from untreated Raji cells to visualize DNA 5′-ends. Exponentially growing Raji cells were harvested, genomic DNA was purified, and LMPCR was performed using the same sets of primers as for the genomic footprints. On the lower strand 5′-ends were found mainly at nonamers B and C, on the upper strand mainly at nonamers A and B (Fig. 3, lanes N). The pattern of 5′-ends at the nonamers in untreated DNA is also summarized in Fig. 4. In addition to 5′-ends at the nonamers, 5′-ends were also found upstream of nonamer C for the lower strand (horizontal arrow above C in Fig. 3a) and upstream of nonamer A for the upper strand (two horizontal arrows above A in Fig. 3b) These upstream 5′-ends are less abundant than 5′-ends at the nonamers. They occur at pyrimidine-rich stretches of the respective strands. Since aphidicolin blocks DNA chain elongation (Huberman, 1981Huberman J.A. Cell. 1981; 23: 647-648Abstract Full Text PDF PubMed Scopus (315) Google Scholar), we did the same experiment on genomic DNA from Raji cells treated with aphidicolin. In this experiment the signal for 5′-ends at the nonamers increased strongly in a time dependent fashion (Fig. 3, lanes 24) (only data for 24 h shown). In addition to this strong signal increase at the nonamers, there was also an increased background and an increased abundance of 5′-ends downstream of nonamer C on the upper strand (nicked arrow below nucleotide 9118 in Fig. 3b). These 5′-ends cannot be readily explained by some mechanism, but they may correspond to damaged DNA, resulting from the prolonged treatment of cells with aphidicolin. The increased background of lanes 24 may also result from DNA damage.Figure 3:Visualization of 5′-ends on both strands of DS in genomic DNA from Raji cells by LMPCR. Panel a shows data for the lower strand, panel b for the upper strand. Lanes A+G and G refer to Maxam and Gilbert, 1980Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 497-560Crossref PubMed Scopus (4) Google Scholar sequencing reactions. Lanes N show DNA 5′-ends occurring in nontreated, nonsynchronously growing cells. Lanes 24 show accumulated 5′-ends in cells treated with 30 μM aphidicolin for 24 h. Experiments were done at least twice with the same results. The numbers on the sides of each panel refer to the EBV sequence, locations of nonamers are indicated by the letters A, B, and C. Horizontal arrows show 5′-ends in genomic DNA from nontreated cells, the nicked arrow indicates an accumulation of 5′-ends in genomic DNA from aphidicolin-treated cells.View Large Image Figure ViewerDownload Hi-res image" @default.
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