Biology subjects

Hyrien, O.

Publications and source records attributed to Hyrien, O..

3 recordsLinked to original sources

bioRxiv2018

Mapping DNA replication with nanopore sequencing

We have harnessed nanopore sequencing to study DNA replication genome-wide at the single-molecule level. Using in vitro prepared DNA substrates, we characterized the effect of bromodeoxyuridine (BrdU) substitution for thymidine on the MinION nanopore electrical signal. Using a neural-network basecaller trained on yeast DNA containing either BrdU or thymidine, we identified BrdU-labelled tracts in yeast cells synchronously entering S phase in the presence of hydroxyurea and BrdU. As expected, the BrdU-labelled tracts coincided with previously identified early-firing, but not late-firing, replication origins. These results open the way to high-throughput, high-resolution, single-molecule analysis of DNA replication in many experimental systems.

genomics↗

bioRxiv2018

Universal temporal rate of DNA replication origin firing: A balance between origin activation and passivation

The time-dependent rate I(t) of origin firing per length of unreplicated DNA presents a universal bell shape in eukaryotes that has been interpreted as the result of a complex time-evolving interaction between origins and limiting firing factors. Here we show that a normal diffusion of replication fork components towards localized potential replication origins (p-oris) can more simply account for the I(t) universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring p-oris. We predict the I(t) maximal value to be the product of the replication fork speed with the squared p-ori density. We show that this relation is robustly observed in simulations and in experimental data for several eukaryotes. Our work underlines that fork-component recycling and potential origins localization are sufficient spatial ingredients to explain the universality of DNA replication kinetics.

biophysics↗

bioRxiv2017

High-throughput optical mapping of replicating DNA

DNA replication is a crucial process for the universal ability of living organisms to reproduce. Existing methods to map replication genome-wide use large cell populations and therefore smooth out variability between chromosomal copies. Single-molecule methods may in principle reveal this variability. However, current methods remain refractory to automated molecule detection and measurements. Their low throughput has therefore precluded genome-wide analyses. Here, we have repurposed a commercial optical DNA mapping device, the Bionano Genomics Irys system, to map the replication signal of single DNA molecules onto genomic position at high throughput. Our methodology (HOMARD) combines fluorescent labelling of replication tracks and nicking endonuclease (NE) sites with DNA linearization in nanochannel arrays and dedicated image processing. We demonstrate the robustness of our approach by providing an ultra-high coverage (23,311 x) replication map of bacteriophage {lambda} DNA in Xenopus egg extracts. HOMARD opens the way to genome-wide analysis of DNA replication at the single-molecule level.

genomics↗