Abstract
Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons.
Highlights
Genomic DNA is duplicated during the S-phase of the eukaryotic cell cycle
The absence of phototoxicity-derived effects was supported by two lines of evidence: first, cells commonly entered into mitosis after being illuminated for the whole-cell cycle (Fig. 1a and Supplementary Movie 1); and second, the cell cycle duration (22.6 h) measured from microscopic images of live cells and the time needed for the culture to double in the absence of illumination were essentially the same (Fig. 1b and Table 1)
Cells with uniformly distributed nuclear PCNA foci were classified as being in early S-phase (Se), perinucleolar foci rings were used as main marker of mid S-phase cells (Sm) and bright replication foci (RFi) clusters were used to distinguish cells in late S-phase
Summary
Genomic DNA is duplicated during the S-phase of the eukaryotic cell cycle. At the chromatin fibre level, DNA replication can be characterized by the location on the DNA molecule where the DNA synthetic complexes (replisomes) are assembled and replication is initiated (the so-called origin of replication) and by the actual positions where DNA synthesis occurs at any given moment, termed replication forks[1]. Size and the rate of replication fork movement, were originally obtained from pattern analysis of tritiated thymidine-labelled tracks of replication forks on extended DNA molecules[5,6,7]. These DNA autoradiography findings suggested that the genome replicates via clusters of small (50–300 kbp) synchronously activated replicons[8,9]. The stability of RFi over several cell cycles and characteristics of their brightness suggested a relation of nuclear RFi to tandem clusters of synchronously activated replicons described on DNA fibres[12]. On the basis of these initial studies, RFi were for decades considered as complex functional–structural units of chromatin that contained multiple replicons[26,27]
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