4D Visualization of replication foci in mammalian cells corresponding to individual replicons

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.

Nucleoporins redistribute inside the nucleus after cell cycle arrest induced by histone deacetylases inhibition

“…By confocal microscopy, PCNA label in TSA treated cells exhibited a diffuse pattern, typical for G0/G1 cells. 48 As expected, Nup153 formed INCs, but they did not colocalize with any appreciable structure of PCNA. Similar results were obtained after double staining of Nup153 with RNApol II Ser5P.…”

“…Colorless nuclei correspond to cells in early G1, which are starting to synthesize Cdt1-RFP.. Yellow nuclei belong to cells at the G1/S transition, when Cdt1-RFP is starting to be degraded while Geminin-EGFP is already transcription factories [45][46][47] and replication foci. 48,49 Both processes might be altered when HDACi are present, due to its chromatin decondensation properties and cell cycle arrest effect. As some nucleoporins are proposed to interact with active transcribed chromatin and assist DNA replication, 18,50,51 we hypothesized that INCs might be enriched in markers of such nuclear structures.…”

A journey through the microscopic ages of DNA replication

“…These combined efforts led to a further increase in the numbers of replication sites measured in cells (see Fig. 3), which was now finally compatible and fitting with the predicted numbers of replicons needed to duplicate the genome in human cells (Chagin et al 2016; Löb et al 2016). …”

“…Previous efforts by Shaw et al (2010), together with measurements throughout the years culminating on the visualization and quantification of individual replicons in cells in 4D, all supported by 3D–SIM imaging (Chagin et al 2016) were all combined in a minimalistic but comprehensive 4D replicon simulation model (Löb et al 2016) displaying previously published replication polarity gradients, replication timing profiles, N/U domains, topologically associating domains, and timing transition regions (Audit et al 2013; Baker et al 2012; Chen et al 2010; Hyrien et al 2013; Pope et al 2014). …”

“…Each replicon is replicated by two replication machineries ( magenta ), composed of various replication proteins, magnified in the bottom left corner . Adapted from (Chagin et al 2016; Chagin et al 2010) …”