Roxane Lestini Laboratoire d’Optique et Biosciences, École Polytechnique

Archaea provide a simplified yet highly informative model for deciphering the complex regulatory networks governing DNA replication and origin firing. For example, Haloferax volcanii has a chromosome with four active replication origins distributed across a 3.5 Mb DNA molecule and exhibit some distinctive properties, such as a heterogeneous polyploidy, carrying 10–18 copies of its genome. To investigate DNA replication in this organism, we combined complementary approaches: Marker Frequency Analysis by sequencing (MFA-seq) to capture the global replication profile at the population level, super-resolution STORM microscopy to visualize replication forks and assesses replication diversity at the single-cell level, and very recently fluorescence in situ hybridization (FISH) to detect specific genomic regions to explore ploidy variation and correlate it with the number of replication foci at the single-cell level. These complementary approaches yield consistent results, providing a coherent picture of the replication dynamics. Furthermore, our findings suggest that under conditions where the generation time is increased (e.g., at 37°C), there is a significant reduction in ploidy. By integrating these techniques, we aim to characterize the replication program in H. volcanii and gain a deeper understanding of its regulation.