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First published online September 2, 2003
doi: 10.1242/10.1242/jcs.00708

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Complex protein-DNA dynamics at the latent origin of DNA replication of Epstein-Barr virus

¹ Department of Gene Vectors, GSF-National Research Center for Environment and Health, Marchioninistrasse 25, 81377 München, Germany
² Institute for Immunology, GSF-National Research Center for Environment and Health, Marchioninistrasse 25, 81377 München, Germany

View larger version (41K): [in a new window]   Fig. 1. Cell-cycle-dependent chromatin-binding of proteins in A39 cells. Cell cycle phases of logarithmically growing A39 cells were separated by centrifugal elutriation. The DNA content of the different fractions was determined by FACS. Soluble and chromatin-bound proteins of each fraction were separated by cell fractionation and investigated after SDS-PAGE by immunoblot analysis. (A) FACS profiles of the separated fractions (top) and immunoblot analysis of chromatin-associated cyclins A, B1 and E (bottom). (B) Immunoblot studies of EBNA1 and pre-RC components using antibodies as indicated on the right. Chromatin-binding experiments of hsOrc1 were performed in parallel in the presence or absence of 25 µM MG132 as indicated on the left. (Bottom) To analyse the relevance of a 26S-proteasome-dependent degradation, whole cell extracts (WCE) were prepared by lysing cells in RIPA buffer in the absence (left) or presence (right) of 25 µM MG132. After SDS-PAGE of equivalent amounts of WCE, the presence of HsOrc1p was detected by immunoblotting. (C) Relative ratios of chromatin bound proteins analysed in (B). Signal intensities of the respective autoradiograph were quantified using NIH Image and plotted against the flow rate (ml minute-1) corresponding to cell cycle progression. The highest intensity of each individual factor was set to 100%. HsOrc4p and HsOrc6p are not shown for the clarity of the figure. (D) For G0 experiments, A39 cells were grown to high density and arrested for 3 days. G0-arrested and logarithmically growing A39 cells (FACS profiles on the top left) were fractionated using the chromatin-binding protocol. Soluble (S) and chromatin-bound (Ch) proteins were separated by SDS-PAGE and immunoblots were probed with antibodies as indicated on the right.

View larger version (26K): [in a new window]   Fig. 2. EBNA1 binds cell cycle independently at oriP. (A) Mini-EBV plasmid 1478.A used to immortalize human primary B cells. Some functional elements are shown on the outer circle. The plasmid backbone derived from the F-factor plasmid pMBO132 (arrows). EBNA1 is shown with some cis elements [OriP, oriLyt (the lytic origin of DNA replication), the terminal repeats and the W repeats]. The inner circle of the map indicates the locations of the fragments that were analysed by PCR amplification after immunoprecipitation. (B) Enlarged view of oriP (top). The locations and designation of the PCR fragments used to scan the binding sites of EBNA1, HsOrc and Mcm2-Mcm7 proteins are shown below the ruler (sc2-sc10, I3). Different cell cycle phases were separated by centrifugal elutriation and five cell cycle fractions were subjected to ChIP (G1, 40 ml minute-1; G1/S, 50 ml minute-1; S, 60 ml minute-1; S/G2, 80 ml minute-1; G2/M, 90 ml minute-1). Cross-linked chromatin of 1x107 cells was used for each immunoprecipitation. Co-precipitated DNA was isolated and 1/50 thereof was used for each PCR. The histogram shows the result of EBNA1-experiments. The difference between the crossing points of the EBNA1 immunoprecipitate and the isotype control is indicated on the logarithmic y axis (Cp). The threshold level marked by the reference I3 is indicated as dotted line. The graph shows the mean values and standard deviations from three independent experiments.

View larger version (34K): [in a new window]   Fig. 5. OriP-bound proteins in G0-arrested cells. The same G0 and logarithmically growing cells as analysed in Fig. 1D were used to determine oriP-bound proteins. The ChIP experiments were performed and analysed using antibodies directed against HsOrc1p, HsOrc2p, HsOrc3p, HsOrc6p, HsMcm7p and EBNA1. The mean values and standard deviations are calculated from seven independent experiments.

View larger version (34K): [in a new window]   Fig. 3. HsOrc binding at oriP during the cell cycle. Different cell cycle phases were separated by centrifugal elutriation and five cell cycle fractions were subjected to ChIP (G1, 40 ml minute-1; G1/S, 50 ml minute-1; S, 60 ml minute-1; S/G2, 80 ml minute-1; G2/M, 90 ml minute-1). Cross-linked chromatin of 1x107 cells was used for each immunoprecipitation with antibodies directed against HsOrc3p (A), HsOrc6p (B) and HsOrc1p (C). Co-precipitated DNA was isolated and 1/50 thereof was used for each PCR. The mean values and standard deviations are calculated of four (A), and seven independent experiments (B,C).

View larger version (33K): [in a new window]   Fig. 4. Cell-cycle-dependent binding of HsMcm2p-HsMcm7p at oriP. Different cell cycle phases were separated by centrifugal elutriation and five cell cycle fractions were subjected to ChIP (G1, 40 ml minute-1; G1/S, 50 ml minute-1; S, 60 ml minute-1; S/G2, 80 ml minute-1; G2/M, 90 ml minute-1). Cross-linked chromatin of 1x107 cells was used for each immunoprecipitation. Co-precipitated DNA was isolated and 1/50 thereof was used for each PCR. Antibodies directed against HsMcm3p (A) and HsMcm7p (B) were used to visualize the cell-cycle-dependent binding of this complex. The values are calculated from three independent experiments.

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