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First published online 5 October 2004
doi: 10.1242/jcs.01412

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Stable chromosomal units determine the spatial and temporal organization of DNA replication

¹ University of Munich (LMU), Department Biology II, Grosshaderner Str. 2, 82152 Planegg-Martinsried, Germany
² Max Delbrück Center of Molecular Medicine, Wiltbergstr. 50, 13125 Berlin, Germany
³ European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany

View larger version (63K): [in a new window]   Fig. 1. Overall nuclear dynamics during S-phase progression in living HeLa cells. (a) Labelling scheme. (b-d) Time series (time points indicated in minutes after microinjection) of a double-labelled nucleus (green, GFP-PCNA; red, Cy3). Colocalizing Cy3 and GFP fluorescence appears yellow. Each panel displays a projection of four focal planes (z=600 nm). The GFP-PCNA pattern proceeds from a mid (45 minutes) to a late (330 minutes) S-phase pattern. After initial colocalization of nascent DNA with GFP-PCNA foci (45 minutes), GFP-PCNA foci appear at sites adjacent to nascent DNA at 145 minutes and at increasingly distant sites during S-phase progression (see enlargements of the framed regions in the insets). The large arrowhead indicates a region where GFP-PCNA foci disappeared after DNA synthesis. Two replication sites (small arrows) at the nucleolar (N) periphery are shown enlarged in Fig. 2. Small arrowheads indicate Cy3-labelled cytoplasmic vesicles. Bar, 5 µm; inset bar, 1 µm. (e,f) The distance between the two foci of a given pair of Cy3-labelled DNA foci was measured at two different time points (t=25 minutes). The bars indicate the percentages of pairs of foci showing changes in their distances within a given interval (0-100 nm, 101-200 nm etc.) after 25 minutes. (e) To determine DNA dynamics during replication, it was ensured that at both time points both DNA foci of a given pair colocalized with GFP-PCNA. (f) DNA dynamics after replication of the foci were measured in the same nuclei at later time points, when GFP-PCNA foci occupied different nuclear regions. n, number of pairs of foci evaluated.

View larger version (97K): [in a new window]   Fig. 2. Local dynamics at DNA replication sites in living HeLa cells. (a) Enlargements of two replication sites associated with the nucleolar (N) periphery (labelled with small arrows in Fig. 1) at the indicated time points (minutes after microinjection). GFP and Cy3 fluorescence and the corresponding merged images are shown (green, GFP; red, Cy3). At 95 minutes the nascent DNA synthesized at the two GFP-labelled sites is translocated between these two sites (arrow). At 120 minutes intense GFP fluorescence appears at a site previously devoid of GFP-PCNA (arrowhead), adjacent to a previous site of synthesis where the nascent DNA is still associated with the nucleolar periphery. (b) Fluorescence intensity profiles along the lines indicated on the merged images in a. The x-axis indicates the path of the line measured in µm with 0 corresponding to the left end of the line. Arrow and arrowhead point to the same regions as in a. Bars, 1 µm.

View larger version (103K): [in a new window]   Fig. 3. DNA shows only local rearrangements whereas the replication machinery appears at neighbouring sites. The nucleus was labelled according to the labelling scheme depicted in Fig. 1a. (a) Time series (minutes after microinjection) of a perinucleolar region. The nucleolus appears dark and is labelled N. Projections representing four consecutive light optical sections (z=300 nm) are depicted for each time point. The merged images of GFP (green) and Cy3 fluorescence (red) are shown with colocalization appearing yellow. The upper three panels represent the whole area whereas enlargements of two regions of nascent DNA synthesis labelled 1 and 2 are displayed in the lower six images. A large arrowhead indicates the first site of synthesis at region 1. A small arrowhead points to a GFP-labelled focus appearing in a closely adjacent region first visible at 58 minutes. Nascent DNA extruded away from the nucleolar border at 58 minutes at site 2 is labelled with a large arrow (the GFP-labelled focus at site 2 remains associated with the nucleolar periphery). At 82 minutes all nascent DNA is again closely associated with the nuclear periphery. In regions at the nucleolar periphery so far devoid of nascent DNA, new GFP-PCNA foci appeared. A small arrow indicates a GFP-labelled focus that appeared at 82 minutes in such a region devoid of nascent DNA. (b) Fluorescence intensity scans along the lines indicated on the images in a (arrowheads and arrows point to the same regions as in a), with 0 µm corresponding to the left end of the line in a. (c) Consecutive light optical sections (confocal raw data, z=300 nm) from the enlarged region at 32 minutes and at 58 minutes (arrowheads as in a and b). The new GFP-labelled focus appearing at the left of site 1 at 58 minutes did not relocalize to this site from another nuclear plane. (d) Projections of three consecutive light optical sections from the same perinucleolar area at 348 minutes after microinjection. The nucleus progressed normally into G2. During G2, GFP-PCNA is uniformly distributed over the extranucleolar nuclear regions. The nucleolus appears dark and the Cy3-labelled DNA is still associated with the nucleolar periphery. Bars, 1 µm.

View larger version (36K): [in a new window]   Fig. 4. Experimental procedure and selection of daughter cells for imaging. (a) Scheme depicting the experimental procedure. Cells were microinjected during the first S phase with a mixture of the GFP-PCNA expression plasmid and Cy3-dUTP. After 1.5 hours, some cells were microinjected again with Cy5-dUTP. Cells were imaged during the next S phase after cell division. (b) Schematic drawing of nuclei selected for imaging. During the imaging period following cell division, labelled cells were at different cell cycle and S-phase stages. Cells not in S phase at all (left, uniform distribution of GFP-PCNA) or not at that stage of S phase that corresponded to the DNA-labelling pattern (middle, GFP-PCNA localizing in different nuclear regions to the labelled DNA) were not chosen for imaging. Only those cells where GFP-PCNA foci and labelled DNA foci occupied similar nuclear regions were imaged (right).

View larger version (38K): [in a new window]   Fig. 5. Spatial dynamics of GFP-PCNA at sites harbouring labelled DNA foci. (a) Light optical section of a nucleus labelled according to the scheme depicted in Fig. 4a at the beginning of the imaging period (red, Cy3 fluorescence; green, GFP fluorescence; yellow, colocalization). The arrow points to a region shown enlarged in b-d. (b-d) Time series (indicated in minutes) of the nuclear sub-region marked with an arrow in a. The arrow in b points to a Cy3-labelled focus that colocalized with a GFP-PCNA focus at the beginning of the imaging period. At later time points (c,d), the GFP fluorescence appeared in an adjacent region (arrowhead) between this and a neighbouring Cy3-labelled focus. Bar in a, 5 µm; b-d, 1 µm.

View larger version (37K): [in a new window]   Fig. 6. Analysis of Cy3 and GFP-PCNA colocalization patterns. (a) A scheme summarizing the spatiotemporal dynamics observed. During the temporal progression of S phase (indicated by black arrows) DNA foci (red) stably occupy their nuclear positions, whereas GFP-PCNA foci appear at adjacent sites. A transition stage is observed during which GFP foci at the previously active sites cease, whereas GFP foci at adjacent new sites increase (compare with Fig. 3a and b, 58 minutes and 82 minutes). Colocalization with labelled DNA (middle) is only observed during a restricted period (compare Figs 2 and 5). This scheme illustrates the behaviour of the majority of replication sites and does not take into account intranuclear heterogeneity, which might occur when the replication programme is not exactly reproduced at all sites. (b-d) Quantitative analysis of colocalization between Cy3 and GFP-PCNA foci in daughter cells. 21872 Cy3-labelled and 17401 GFP-labelled foci were evaluated. Red and yellow bars represent the percentage of Cy3-labelled foci colocalizing (yellow) or not colocalizing (red) with GFP-PCNA labelled foci. Green bars represent the percentage of GFP-labelled foci not colocalizing with Cy3-labelled foci. Individual nuclei display different degrees of colocalization. (b) In class I nuclei on average only about 15% of Cy3-labelled foci colocalize with GFP-labelled foci. (c) In class II nuclei about half of the Cy3-labelled foci colocalize with GFP-PCNA foci. (d) In class III nuclei on average about 85% of Cy3-labelled foci colocalize with GFP-labelled foci.

View larger version (60K): [in a new window]   Fig. 7. Spatial arrangements of GFP-PCNA and Cy3-labelled foci in class I and class III nuclei. Nuclei were labelled according to the experimental procedure outlined in Fig. 4a. GFP and Cy3 fluorescence and the corresponding merged images (green, GFP; red, Cy3; yellow, colocalization) are shown. (a) Single light optical sections of a class I nucleus. The upper panels show the nuclear periphery whereas the lower panels show an equatorial plane. GFP-PCNA foci and labelled DNA foci localize adjacent to each other. (b) Single light optical sections showing an equatorial plane of a class III nucleus. The nucleus displays a pattern typical for early S phase with many foci distributed throughout the nuclear interior. Colocalization at the level of individual foci becomes evident in regions relatively devoid of labelled foci (arrows). However, colocalization at the level of individual foci can also be observed in more crowded regions (arrowheads). Bars, 10 µm.

View larger version (98K): [in a new window]   Fig. 8. Spatiotemporal interaction of the replication machinery with adjacent sets of DNA foci. (a) The panels show light optical sections from the equatorial planes of two sister nuclei labelled and imaged according to the experimental procedure outlined in Fig. 4a. Arrows point to regions with labelled foci shown enlarged in b. The panels show single GFP, Cy3 and Cy5 fluorescence with merged image as indicated (green, GFP; red, Cy3; blue, Cy5; red and green appears yellow; red and blue appears violet; a merge of all three colours appears white). The number of GFP foci corresponds to the number of Cy3-labelled foci. (b) GFP-labelled foci colocalize with Cy3 but not with adjacent Cy5-labelled foci. Bar in a, 10 µm; b, 1 µm.

View larger version (37K): [in a new window]   Fig. 9. Spatial arrangements of GFP-PCNA foci and labelled DNA foci in triple-labelled daughter cells. Equatorial planes of two nuclei labelled and imaged according to the experimental procedure outlined in Fig. 4a are shown. GFP, Cy3 and Cy5 fluorescence and the merged images are shown as indicated (green, GFP; red, Cy3; blue, Cy5; red and green appears yellow; red and blue appears violet; green and blue gives rise to cyan; and a merge of all three colours appears white). (a,b) A daughter nucleus at an earlier stage of S phase during labelling than the mother cell, indicated by relatively numerous GFP-PCNA foci occupying interior positions. The boxed regions are shown enlarged in b. GFP-PCNA foci are adjacent to Cy3-labelled but not to Cy5-labelled DNA foci. (c,d) A nucleus at the transition to a later stage of S-phase than the mother cell during labelling, indicated by the relatively low number of GFP-foci compared to the number of Cy3- or Cy5-labelled foci. The framed regions are shown enlarged in d. (d) GFP-PCNA foci colocalize either with Cy5-labelled foci (upper panels) or sites adjacent to Cy5-labelled foci (lower panels). Bars in a and c, 10 µm; in b, 1 µm; in d, 0.5 µm.

View larger version (36K): [in a new window]   Fig. 10. Models of DNA replication. (a) The replication machinery follows an established path of sub-chromosomal foci during the temporal progression of S phase. The scheme depicts a cell nucleus harbouring a nucleolus (N) and DNA organized into sub-chromosomal foci. The spatial organization of sub-chromosomal foci with a defined replication timing (red, early; blue, mid; black, late) is established during G1 and is stably maintained during interphase (see Sadoni et al., 1999). Individual chromosome territories organized into sub-chromosomal foci are shown on the left. For simplicity, only a limited number of individual territories are shown, each harbouring only a few sub-chromosomal foci (it is estimated that sub-chromosomal foci harbour a DNA content of about 1 Mb). During S-phase progression, the replication machinery (green stars indicate replication foci, GFP-PCNA is uniformly distributed during G1) follows the established pattern of sub-chromosomal foci and neighbouring foci become sequentially occupied. At the level of the whole nucleus (on the right), this results in the typical spatiotemporal patterns of replication foci described in the literature (see Nakayasu and Berezney, 1989; O'Keefe et al., 1992; Sadoni et al., 1999). (b) Substructure of replication foci and activation of neighbouring foci. We propose that replication foci and sub-chromosomal foci correspond to replicon clusters, organized into loops. Furthermore, we propose that the bases of these loops correspond, at least during S phase, to the origins of replication (red), attached to a central structure (blue). Neighbouring foci are connected by one half of a bidirectional replicon. (1) Replication of an earlier replicating focus (left) started, while the neighbouring focus (right) is still inactive. Duplicated DNA is shown in green and the direction of movement of the fork is indicated by green arrows. Unduplicated DNA is shown in black. (2) Replication of the earlier replicating focus finishes and the incoming fork from the earlier replicating focus activates the later replicating neighbouring focus. Synchronous activation (orange star) of the origins of the corresponding replicon cluster might be facilitated by their spatial clustering. (3) Replication of the later replicating focus started.

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