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doi: 10.1242/10.1242/jcs.00087

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The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells

Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260, USA

View larger version (92K): [in a new window]   Fig. 7. The numerous pRb granules are not preferentially associated with perinucleolar regions and do not colocalize with CldU-labeled replication sites during any stage of S-phase. An asynchronous culture of WI38 cells was pulse-labeled for 5 minutes with 10 µM CldU, fixed with formaldehyde and stained with anti-pRb rabbit polyclonal antibodies and anti-nucleolin mouse monoclonal antibodies. The cells were briefly fixed again with formaldehyde before mild HCl hydrolysis and labeling of CldU-substituted DNA with rat monoclonal anti-BrdU antibodies. DNA was stained with DAPI. Images corresponding to double- and triple-labeling protocols were pseudocolored as indicated within each panel. Sites that co-localize appear yellow for red/green, light blue or blue-green for blue/green, and magenta for blue/red merged images, respectively. Based on the appearance of the CldU replication patterns, the cells were classified into early-S (A), mid-S (B) and late-S (C) categories. Besides the strong nucleolar signal, nucleolin exhibits a weaker diffuse nucleoplasmic staining within formaldehyde-fixed cells. This soluble nucleoplasmic form of nucleolin can be removed completely by mild extraction with Triton X100 prior to fixation (not shown). Note the nearly complete lack of colocalization (yellow color) between the pRb (red) and CldU (green) signals in the double-labeled images.

View larger version (70K): [in a new window]   Fig. 8. pRb does not colocalize with Mcm proteins within licensed chromatin in permeabilized cells. An aliquot of the same CldU-labeled W138 asynchronous cell culture used for the results shown in Fig. 7 was extracted for 3 minutes on ice with 0.5% Triton X100 in cytoskeleton (CSK) buffer. The cells were then fixed with formaldehyde and stained with anti-pRb rabbit polyclonal antibodies, anti-Mcm7 mouse monoclonal antibodies and anti-BrdU rat monoclonal antibodies as described in Fig. 7. The cells were classified into G1, early-S, middle-S and late-S according to the presence of detergent-resistant Mcm7 chromatin association, the presence or absence of CldU staining and the type of CldU replication pattern. DNA was stained with DAPI. Images corresponding to double- and triple-labeling protocols were pseudocolored as indicated within each panel. Sites that co-localize appear yellow for red/green, light blue or blue-green for blue/green, and magenta for blue/red merged images, respectively. Identical results were obtained with anti-Mcm2 and anti-Mcm3 antibodies. Compared to Fig. 7, longer exposure times were used to capture the pRb images due to the weaker fluorescent signal as a result of the release of a fraction of nuclear pRb by the nonionic detergent extraction procedure.

View larger version (87K): [in a new window]   Fig. 9. pRb and Mcm proteins are not present at the same nuclear sites within intact cells. An aliquot of the same CldU-labeled WI38 asynchronous cell culture was stained with anti-pRb, anti-Mcm7 and anti-BrdU antibodies as described in Fig. 7 without a prior Triton X100 extraction. Cells were categorized according to the type of CldU replication pattern. Images corresponding to double- and triple-labeling protocols were pseudocolored as indicated within each panel. Sites that co-localize appear yellow for red/green, light blue or blue-green for blue/green, and magenta for blue/red merged images, respectively. Note the presence of Mcm proteins throughout the nucleus at all cell cycle stages.

View larger version (41K): [in a new window]   Fig. 1. The distribution of nuclear DNA replication sites in primary, immortalized and transformed mammalian cells follows identical spatio-temporal patterns during S-phase. Cell cultures synchronized at different stages of S-phase after release from aphidicolin block [5 min (G1/S), 1 hour, 2 hours and 4 hours (early-S), 6 hours and 8 hours (mid-S), 10 hours and 12 hours (late-S)] were pulse-labeled for 5 minutes with 30 µg/ml BrdU, fixed and sites of BrdU incorporation were detected with a nuclease/anti-BrdU cocktail. Based on the number, size, shape and distribution of fluorescent foci, the replication sites were classified into five major types of patterns, which were similar in all cell lines.

View larger version (43K): [in a new window]   Fig. 2. HCl hydrolysis (gray columns) and nuclease digestion (black columns) techniques for visualization of BrdU-labeled nascent DNA reveal identical frequency of the five major replication patterns within S-phase nuclei of exponentially growing primary and immortalized mammalian cells. Shown are results from a single experiment. Identical results were obtained in three independent experiments.

View larger version (62K): [in a new window]   Fig. 3. Spatial organization of DNA replication sites in primary, immortalized and transformed mammalian cells detected by limited HCl depurination of BrdU-substituted DNA. Aliquots of cells from the same synchronized cell populations as in Fig. 1 were used.

View larger version (94K): [in a new window]   Fig. 4. Visualization of replication sites within permeabilized primary or transformed human cells after nuclear run-on replication. (A) Controlled permeabilization with digitonin produces cells with intact nuclei (impermeable to large molecules that lack nuclear localization signal (e.g. Texas Red IgG, right panel) and well preserved nuclear structure. Intact nuclei prepared from WI38 (B) or HeLa (C) cells were introduced into in vitro replication cocktail and nascent DNA at replication forks generated in vivo was labeled with biotin-dUTP. Nuclei were fixed with 70% ethanol and biotin-dUTP was detected with Texas Red-conjugated streptavidin. DNA was stained with 0.1 µg/ml 4',6-diamidino-2-phenylindole (DAPI).

View larger version (53K): [in a new window]   Fig. 5. The replication fork proteins RPA and PCNA, but not nuclear lamins, colocalize with BrdU-labeled nascent DNA within replication foci. HeLa and WI38 cells were pulse-labeled with BrdU, extracted with Triton X100 to remove soluble proteins and fixed with freshly prepared formaldehyde. (A-C) One aliquot of the cells was treated with HCl and then stained simultaneously with lamin B- (green) and BrdU-specific (red) antibodies. A second aliquot was first stained for lamin B [green (D-G)] or lamin A/C [red (J-Q)], the cells were fixed again with formaldehyde, then treated with HCl and stained for BrdU (red in D-F and green in J-Q). (H) The same protocol was used to stain for PCNA (red) and BrdU (green). (I) A fourth aliquot of cells was stained directly with anti-PCNA (red) and anti-RPA (green) antibodies. Results obtained with HeLa cells are shown in panels A-I. Identical results were obtained with WI38 cells.

View larger version (51K): [in a new window]   Fig. 6. pRb is present at hundreds of sites scattered throughout mammalian nuclei. An asynchronous culture of WI38 cells was pulse-labeled for 5 minutes with 30 µg/ml BrdU, fixed with freshly prepared formaldehyde and stained with pRb-specific rabbit polyclonal antibodies (A,D,G) and a nuclease/anti-BrdU labeling cocktail (B,E). DNA was stained with DAPI. (H) pRb foci (red) density is higher within nuclear regions with lower density of DNA (blue).

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