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First published online 28 September 2004
doi: 10.1242/jcs.01405

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Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle

¹ Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
² Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy
³ NEST/INFM and Scuola Normale Superiore, Piazza dei Cavalieri 7, 56126 Pisa, Italy

View larger version (23K): [in a new window]   Fig. 1. Chromatin immunoprecipitation (ChIP) analysis of HeLa cells transiently transfected with Orc1-Flag. (A) Schematic representation of the genomic region comprising the origin of DNA replication associated to the lamin B2 and TIMM 13 genes. The origin sequence is recognized by B48 primers. The control B13 region is also indicated. (B) Crosslinked chromatin was immunopurified with anti-Flag (-Flag) or with a control non-specific antibody (-IgG). After reversion of crosslinking, immunopurified DNA was subjected to competitive PCR with B48 and B13 primer sets. Dilutions of the competitor DNA (see Materials and Methods) are indicated above each lane. The identity of the amplification bands is indicated on the right of each panel. The relative abundance of B48 and B13 sequences in the immunopurified DNA is determined by the ratio of genomic (G) to competitor (C) bands. (C) Histogram showing the enrichment of B48 over B13 sequences in the immunopurified DNA obtained with -Flag and -IgG antibodies. Bar indicates the variation seen in three independent experiments.

View larger version (73K): [in a new window]   Fig. 2. Distribution of Orc1p* in different mammalian cell lines. (A) Exponentially growing human HeLa cells were transfected with either Orc1-Flag or Orc1-GFP. After 48 hours, cells were fixed and processed for conventional microscopy. The distribution of Orc1-Flag was revealed with the rabbit anti-Flag antibody and a TRITC-conjugated anti-rabbit secondary antibody. The localization of Orc1-GFP was revealed by GFP fluorescence. Cells were counterstained with DAPI. The distribution of Orc1-GFP was also determined in transfected monkey COS7 and mouse NIH-3T3 cells. (B) Asynchronous NIH-3T3 cells were transfected with Orc1-Flag and after 48 hours were fixed and stained with the rabbit anti-Flag and the rat anti-HP1ß antibodies. Antigen-antibody complexes were revealed with a TRITC-conjugated anti-rabbit secondary antibody and with a Cy5-conjugated anti-rat secondary antibody (visualized in green). Nuclei were stained with DAPI. Confocal images of the same field were taken. Bar, 10 µm.

View larger version (35K): [in a new window]   Fig. 3. Orc1p* labels G1 cells. (A) HeLa cells expressing Orc1-GFP were stained with the anti-PCNA PC10 monoclonal antibody and with the anti-mouse Cy5-conjugated secondary antibody (red). The fluorescent signal of the Orc1-GFP fusion (green) is also shown. Confocal laser images of the same field were taken and merged. The arrow indicates a PCNA-negative nucleus expressing Orc1-GFP. Bar, 10 µm. (B) FACS analysis. The FACS profile in the left side panel shows the fluorescence of the total cell population 48 hours after transfection. The gate (dotted line) shows the sub-population of transfected cells expressing Orc1-GFP (29% of total). The other two panels show the DNA content of the total cell population (ALL) and of cells expressing Orc1-GFP (GATED). The distribution of the cells in the cell cycle according to the DNA content is indicated on the right side of each panel.

View larger version (46K): [in a new window]   Fig. 4. Orc1p* in S-phase nuclei. HeLa cells were transfected with a plasmid that directs the expression of a dicistronic mRNA in which the sequence of Orc1-Flag is upstream of the GFP. (A) Schematic diagram of the pOrc1-Flag/GFP plasmid. PCMV, cytomegalovirus promoter; IRES, internal ribosomal entry site; SV40 pA, SV40-derived RNA processing site. (B) Cells were co-stained with the anti-Flag polyclonal and anti-PCNA monoclonal antibodies. Antibodies were revealed with the TRITC-conjugated anti-rabbit and Cy5-conjugated anti-mouse secondary antibodies. Nuclei were stained with DAPI. Confocal laser images were taken. The overlay of GFP (visualized in green), Orc1-Flag (red) and PCNA (blue) is shown (merge). The arrows indicate PCNA-positive GFP-positive nuclei in which Orc1-Flag is not detectable. Arrowheads show transfected nuclei expressing Orc1-Flag that are PCNA-negative. To preserve the GFP signal, we skipped Triton extraction before fixation. Under these conditions PCNA staining was detectable not only in S-phase replicative patterns but also in G2-phase cells characterized by intense homogenous staining. Bar, 10 µm. (C) Shows a PCNA-positive, Orc1p*-positive nucleus in early S phase (early-S) and a PCNA-positive, Orc1p*-negative nucleus in mid S phase (mid-S). The merged images show that in early S phase, Orc1p* does not colocalize with PCNA.

View larger version (40K): [in a new window]   Fig. 5. Orc1p* in G1-phase nuclei. (a-h) Transiently transfected NIH-3T3 cells were blocked at the mitotic spindle checkpoint with nocodazole and allowed to recover for 2 hours (a-d) and 6 hours (e-h). Fixed cells were incubated with the rabbit anti-Flag polyclonal antibody (a,e), and with the anti-HP1ß antibody (b,f). Antibodies were revealed with the anti-rabbit TRITC-conjugated (red) and with the anti-rat Cy5-conjugated (green) antibodies. Confocal laser images are shown. The overlay of Orc1p* (red) and HP1ß (green) is also shown (merge). Chromatin was stained with DAPI (d,h). Bar, 10 µm.

View larger version (25K): [in a new window]   Fig. 6. Analysis of Orc1p mutants. (A) All mutants were fused to either GFP or Flag-epitope and their subcellular distribution was assessed in transiently transfected NIH-3T3 cells. The region of human Orc1p included in each fusion is indicated by the corresponding amino acids on the left hand side. Internal deletion mutants are indicated with a symbol. Substitution mutants are indicated by the corresponding single-code amino acid letter. Black bars indicate substituted residues. The ability of each mutant to accumulate at DAPI foci was scored as + (targeting proficient) or – (targeting deficient). The localization in cell nucleus (N) or in the cytoplasm (C) is indicated. A diagram including the main functional domains of the protein is shown at the top. BAH, bromo-adjacent-homology domain; HP1, HP1-binding site (Pak et al., 1997); Sir1, Sir1-binding site (Zhang et al., 2002a); AAA, ATPase domain; HW, putative DNA binding site (Liu et al., 2000). (B) Immunolocalization of the 151-269 mutant fused to the Flag epitope in NIH-3T3 cells. Chromatin was stained with DAPI. Confocal laser images of the same field were taken and merged. (C) Immunolocalization of the A264,265,266 mutant fused to the Flag epitope in NIH-3T3 cells. Images show cells with cytoplasmic (left) or nuclear (right) distribution of the overexpressed protein. Chromatin was stained with DAPI. Confocal laser images were taken. Bar, 10 µm. (D) Chromatin-binding of Orc1p* (wt) and Orc1-K540T-Flag (K540T) mutant was analyzed by western blotting with the anti-Flag antibody. Transiently transfected HeLa cells were synchronized in G1-phase and fractionated as described in Materials and Methods. The amount of protein in the chromatin (P3) and soluble fractions (S1) was compared for each construct. The amount of P3 fraction analyzed for each sample was corrected for the efficiency of transfection determined by measuring in parallel the fraction of Orc1p* expressing cells. The level of endogenous Orc2p in the loaded P3 fractions is also shown. TE, total cell extracts. (E) Nuclear extracts (NE) of HeLa cells expressing K540T-Flag epitope (+) and of non-transfected cells (–) were probed with the anti-Flag polyclonal antibody and the anti-Orc2 polyclonal antibody. The same extracts were used in co-immunoprecipitation experiments with the monoclonal anti-Flag M2 affinity gel (IP). The presence of Orc1-Flag and of Orc2p in the immunoprecipitate was revealed by western blotting with anti-Flag and anti-Orc2 polyclonal antibodies.

View larger version (38K): [in a new window]   Fig. 7. Orc1p but not Orc2p binds to HP1. (A) GST pull-down experiment performed by incubating 2 µg of either GST or GST-HP1 immobilized on glutathione-agarose beads with in vitro translated [35S]-labeled Orc1p or Orc2p. After binding at 4°C, the beads were extensively washed and bound proteins were loaded onto a 10% acrylamide-SDS gel. The upper panel shows the autoradiograph; the lower panel shows the gel after staining with Coomassie blue. The input lanes contain the labeled proteins prior to binding. (B) Quantification of the GST pull-down experiment from A. The amount of radioactivity bound to the beads is indicated as a percentage of the input material. (C) HP1 binds to a region of Orc1p encompassing amino acids 151-269. The indicated [35S]-labeled deletion mutants of Orc1p were incubated with either GST or GST-HP1 and processed as described in A. (D) Quantification of the GST pull-down experiment of panel C. (E) Visualization of FRET in human HeLa cells. The plasmids indicated on top of each column were transfected in asynchronous HeLa cells; individual transfected cells were visualized by excitation at 480 nm and collection at 520 nm, showing EGFP fluorescence after direct EGFP excitation (panels in row a), and by excitation at 350 nm and collection at 520 nm, showing EGFP fluorescence after BFP excitation, indicating FRET (panels in row b). (F) Quantification of FRET between EGFP-HP1 and BFP-Orc1. Fluorescent emission at 520 nm from individual cells transfected with the indicated constructs was recorded after excitation at 350 or 480 nm, and integrated intensities over the whole cell were evaluated. The plotted values (indicated by dots) represent the ratio between these two measurements: higher values indicate more efficient resonant energy transfer between BFP and EGFP. Ten consecutively analyzed cells were considered for each transfection; both their individual fluorescence ratios and their percentile box plot distributions are shown. In each box, the horizontal lines from top to bottom mark the 10th, 25th, 75th and 90th percentiles. Cells transfected with pEGFP-HP1 and pBFP-Orc1 plasmids showed FRET between the two fluorescent proteins that was dependent on the presence of both the HP1 and Orc1p moieties, thus indicating binding between these two proteins in vivo.

View larger version (62K): [in a new window]   Fig. 8. Orc1p* focalization survives TSA and RNase treatment. (A) NIH-3T3 cells were treated with 0, 0.1 and 0.5 µg/ml TSA for 6 hours. Fixed cells were stained with the anti-HP1 antibody. Heterochromatin was stained with DAPI. Dispersal of HP1 from DAPI foci is already detectable at 0.1 µg/ml TSA. (B) Cells expressing Orc1-Flag were incubated in the presence of 0.5 µg/ml TSA as in panel A. Fixed cells were co-stained with the anti-Flag and anti-HP1 antibodies. Heterochromatin was stained with DAPI. In contrast to HP1, Orc1-Flag still colocalizes with DAPI foci. (C) Cells expressing Orc1-Flag were incubated with RNase A as described in Materials and Methods. Orc1-Flag was visualized with the anti-Flag antibody; heterochromatin was stained with DAPI visualized in green for a better resolution of the merged image. Confocal laser images are shown. Bar, 10 µm.