Biology subjects

Ciardo, D.

Publications and source records attributed to Ciardo, D..

4 recordsLinked to original sources

bioRxiv2026

High-resolution single-molecule replication profiling of the human genome

Despite the considerable progress made in recent years thanks to the rise of long-read sequencing, high-resolution, single molecule (SM)-based replication profiling of large genomes has remained out of reach. Here, we present a replication labelling strategy consisting in repeated pulse-labelling of asynchronously growing cells with the thymidine analogue bromodeoxyuridine (BrdU) that greatly enhances the number of detectable replication tracks. We show that the multipulse BrdU labelling protocol, in association with ForkML, a machine-learning method translating BrdU signals in nanopore reads into oriented and positioned replication forks, enables the establishment of a SM replication map of the entire human genome at kilobase resolution.

genomics↗

bioRxiv2026

A Closed-Form Bayesian Framework for DNA Replication Reveals Intrinsic Origin Timing and Activation Delays

I.We present an analytical framework for modeling eukaryotic DNA replication that, given experimental Replication Fork Directionality (RFD) data, enables Bayesian inference of origin number, activation delay [Formula] and intrinsic timing ({lambda}i), (the mean replication time if each origin were isolated). By deriving closed-form expressions for RFD and Mean Replication Timing (MRT) under exponential and a specific Weibull firing-time distributions as functions of [Formula] and ({lambda}i), we eliminate the need for stochastic simulations. These analytical results reveal that RFD, as a ratio of fork directions, is invariant under joint rescaling of intrinsic timing and fork speed; absolute intrinsic timing can nonetheless be inferred when fork speed is independently measured. We demonstrate that under exponential firing distribution for the origin, the observed efficiency (Ei), i.e. the probability for an origin to fire which accounts for nearby origin, is simply MRT(xi)/{lambda}i. The closed-form RFD expressions allow to use a Bayesian method that achieves 0.96-0.99 correlation with yeast RFD profiles and resolves ~780 origins in S. cerevisiae. Our framework identifies about 150 origins with biologically significant delays ([≥] 3 minute), revealing regulated activation kinetics undetectable by existing methods. By quantifying how origin intrinsic timing and delays shape replication timing landscapes, this work confirms yeast as a paradigm organism for studying DNA replication control mechanisms.

biophysics↗

bioRxiv2024

Dual DNA Replication Modes: Varying Fork Speeds and Initiation Rates within the spatial replication program in Xenopus

Large vertebrate genomes duplicate by activating tens of thousands of DNA replication origins, irregularly spaced along the genome. The spatial and temporal regulation of the replication process is not yet fully understood. To investigate the DNA replication dynamics, we developed a methodology called RepliCorr, which uses the spatial correlation between replication patterns observed on stretched single-molecule DNA obtained by either DNA combing or high-throughput optical mapping. The analysis revealed two independent spatiotemporal processes that regulate the replication dynamics in the Xenopus model system. These mechanisms are referred to as a fast and a slow replication mode, differing by their opposite replication fork speed and rate of origin firing. We found that Polo-like kinase 1 (Plk1) depletion abolished the spatial separation of these two replication modes. In contrast, neither replication checkpoint inhibition nor Rif1 depletion affected the distribution of these replication patterns. These results suggest that Plk1 plays an essential role in the local coordination of the spatial replication program and the initiation-elongation coupling along the chromosomes in Xenopus, ensuring the timely completion of the S phase.

bioinformatics↗

bioRxiv2020

Organization of DNA replication origin firing in Xenopus egg extracts : the role of intra-S checkpoint

During cell division, the duplication of the genome starts at multiple positions called replication origins. Origin firing requires the interaction of rate-limiting factors with potential origins during the S(ynthesis)-phase of the cell cycle. Origins fire as synchronous clusters which is proposed to be regulated by the intra-S checkpoint. By modelling the unchallenged, the checkpoint-inhibited and the checkpoint protein Chk1 over-expressed replication pattern of single DNA molecules from Xenopus sperm chromatin replicated in egg extracts, we demonstrate that the quantitative modelling of data requires: 1) a segmentation of the genome into regions of low and high probability of origin firing; 2) that regions with high probability of origin firing escape intra-S checkpoint regulation and 3) the variability of the rate of DNA synthesis close to replication forks is a necessary ingredient that should be taken in to account in order to describe the dynamic of replication origin firing. This model implies that the observed origin clustering emerges from the apparent synchrony of origin firing in regions with high probability of origin firing and challenge the assumption that the intra-S checkpoint is the main regulator of origin clustering. Author summaryDNA replication is one of the fundamental cell functions. The genome of eukaryotic organisms is duplicated from multiple positions named replication origins. Single molecule experiments allow to visualise the dynamics of spatio-temporal patterns created during replication process. The dynamic of replication process is regulated by checkpoints. However, the influence and the role of checkpoint regulation in the dynamics of spatio-temporal patterns of DNA replication is not understood. In this work we build a minimal, process-based and data rooted numerical model that allows to decipher the impact of checkpoint regulation on the dynamics of spatio-temporal pattern of DNA replication.

cell biology↗