March 2019: Is the replication timing control constant through S phase?

Before each cell division, the full genome has to be entirely and faithfully duplicated thereby each daughter cell inherits the complete genetic information. This duplication occurs under the control of a highly sophisticated replication program during the restricted time period corresponding to S phase. DNA replication is initiated at a large number of sites, known as origins of replication, on the chromosomes of eukaryotic cells. In one individual cell, only a part of the origins licensed in G1 phase are activated during S phase thus illustrating the flexible origin choice which is directly related to the stochastic nature of the eukaryotic replication program. Another particularity of the program is that origin activation is also subject to temporal regulation. Like this, some domains of hundred kilobases are replicated in early S phase, others are replicated in mid S phase and the remainders in late S phase. This temporal control is very strict. To date, the factors responsible for the establishment, regulation and maintenance of these domains throughout the cell cycle remain largely unknown. As a first step towards a better comprehension of this temporal program, the team of MN Prioleau has investigated whether the stochasticity of the timing program is changing along the S phase.

Legend: (A) Two alleles of a locus are labelled with the Operator/repressor system TetO/TetR-EGFP and visualised as two bright fluorescent spots in the nucleus of each cell. Spots are in green, and chromatin labelled with Hoechst 33342 is in blue. (B) Real-time imaging of individual cells and measurement of spot intensities: illustration of the synchronous replication of both alleles in one cell. The two spots are numbered 1 and 2 to identify the two alleles. Both alleles of the illustrated cell underwent replication between the image acquisition time points at 90 and 95 min, and the allelic asynchrony (AA) is 0. On the right, variation of the intensity of GFP fluorescence for the two spots (blue and green curves), together with replication time (blue and green dotted lines) is shown.

Comparisons of the precise replication times of allelic loci within single vertebrate cells progressing through S phase at six loci replicated from very early to very late have been performed. These analyses revealed that replication timing is strictly controlled for the three loci replicated in the first half of S phase. Out of the three loci replicated in the second part of S phase, two present a significantly more stochastic pattern.

Surprisingly, the locus replicated at the very end of S phase, presents stochasticity similar to those replicated in early S phase. This locus is in close proximity to nuclear lamina. Such an observation is in agreement with previous studies which reported an overlap between Lamin-associated domains and regions that replicate late during S phase. For going one step further in understanding the potential link between proximity to nuclear lamina and late replication, the late locus with higher degree of stochasticity has been investigated. For this late locus, proximity to nuclear lamina proved to be correlated with a more controlled late replication.

In conclusion, the authors suggest that the richness of loci in efficient origins of replication, which decreases from early- to late-replicating regions, and the strength of interaction with the nuclear lamina may underlie the variation of timing control during S phase.

To know more:

Bénédicte Duriez, Sabarinadh Chilaka, Jean-François Bercher, Eslande Hercul and Marie-Noëlle Prioleau. Replication dynamics of individual loci in single living cells reveal changes in the degree of replication stochasticity through S phase. Nucleic Acids Research, 30 March 2019 (gkz220).

Contact: benedicte.duriez(at)ijm.fr and marie-noelle.prioleau(at)ijm.fr, équipe Domaines chromatiniens et Réplication

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