Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming.

Current temporal studies of DNA replication are either low-resolution or require complex cell synchronisation and/or sorting procedures. Here we introduce Nanotiming, a single-molecule, nanopore sequencing-based method producing high-resolution, telomere-to-telomere replication timing (RT) profiles of eukaryotic genomes by interrogating changes in intracellular dTTP concentration during S phase through competition with its analogue bromodeoxyuridine triphosphate (BrdUTP) for incorporation into replicating DNA. This solely demands the labelling of asynchronously growing cells with an innocuous dose of BrdU during one doubling time followed by BrdU quantification along nanopore reads.

Neural network and kinetic modelling of human genome replication reveal replication origin locations and strengths.

In human and other metazoans, the determinants of replication origin location and strength are still elusive. Origins are licensed in G1 phase and fired in S phase of the cell cycle, respectively. It is debated which of these two temporally separate steps determines origin efficiency.

Genome-wide mapping of individual replication fork velocities using nanopore sequencing.

Little is known about replication fork velocity variations along eukaryotic genomes, since reference techniques to determine fork speed either provide no sequence information or suffer from low throughput. Here we present NanoForkSpeed, a nanopore sequencing-based method to map and extract the velocity of individual forks detected as tracks of the thymidine analogue bromodeoxyuridine incorporated during a brief pulse-labelling of asynchronously growing cells. NanoForkSpeed retrieves previous Saccharomyces cerevisiae mean fork speed estimates (≈2 kb/min) in the BT1 strain exhibiting highly efficient bromodeoxyuridine incorporation and wild-type growth, and precisely quantifies speed changes in cells with altered replisome progression or exposed to hydroxyurea.

FORK-seq: Single-Molecule Profiling of DNA Replication.

Most genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200 nucleotide resolution using a nanopore current interpretation tool allowing the quantification of BrdU incorporation. Along pulse-chased replication intermediates from Saccharomyces cerevisiae, we can orient replication tracks and reproduce population-based replication directionality profiles.

Organization of DNA Replication Origin Firing in 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.

FORK-seq: replication landscape of the Saccharomyces cerevisiae genome by nanopore sequencing.

Genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200-nucleotide resolution. By quantifying BrdU incorporation along pulse-chased replication intermediates from Saccharomyces cerevisiae, we orient 58,651 replication tracks reproducing population-based replication directionality profiles and map 4964 and 4485 individual initiation and termination events, respectively.

The eukaryotic bell-shaped temporal rate of DNA replication origin firing emanates from a balance between origin activation and passivation.

The time-dependent rate [Formula: see text] 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 () can more simply account for the [Formula: see text] universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring . We predict the [Formula: see text] maximal value to be the product of the replication fork speed with the squared density.

Chromatin stiffening underlies enhanced locus mobility after DNA damage in budding yeast.

DNA double-strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear.

Inferring the physical properties of yeast chromatin through Bayesian analysis of whole nucleus simulations.

Background: The structure and mechanical properties of chromatin impact DNA functions and nuclear architecture but remain poorly understood. In budding yeast, a simple polymer model with minimal sequence-specific constraints and a small number of structural parameters can explain diverse experimental data on nuclear architecture. However, how assumed chromatin properties affect model predictions was not previously systematically investigated.

Connecting localized DNA strand displacement reactions.

Logic circuits based on DNA strand displacement reactions have been shown to be versatile enough to compute the square root of four-bit numbers. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this paper, we consider simple examples of these circuits when they are attached to platforms (DNA origamis).

How to build a yeast nucleus.

Biological functions including gene expression and DNA repair are affected by the 3D architecture of the genome, but the underlying mechanisms are still unknown. Notably, it remains unclear to what extent nuclear architecture is driven by generic physical properties of polymers or by specific factors such as proteins binding particular DNA sequences. The budding yeast nucleus has been intensely studied by imaging and biochemical techniques, resulting in a large quantitative data set on locus positions and DNA contact frequencies.

Guided assembly of tetramolecular G-quadruplexes.

Nucleic acids are finding applications in nanotechnology as nanomaterials, mechanical devices, templates, and biosensors. G-quadruplex DNA, formed by π-π stacking of guanine (G) quartets, is an attractive alternative to regular B-DNA because of the kinetic and thermodynamic stability of quadruplexes. However, they suffer from a fatal flaw: the rules of recognition, i.

Cooperativity in the annealing of DNA origamis.

DNA based nanostructures built on a long single stranded DNA scaffold, known as DNA origamis, offer the possibility to organize various molecules at the nanometer scale in one pot experiments. The folding of the scaffold is guaranteed by the presence of short, single stranded DNA sequences (staples), that hold together separate regions of the scaffold. In this paper, we modelize the annealing-melting properties of these DNA constructions.

Modeling the mechanical properties of DNA nanostructures.

We discuss generalizations of a previously published coarse-grained description [Mergell et al., Phys. Rev.

Direct visualization of transient thermal response of a DNA origami.

The DNA origami approach enables the construction of complex objects from DNA strands. A fundamental understanding of the kinetics and thermodynamics of DNA origami assembly is extremely important for building large DNA structures with multifunctionality. Here both experimental and theoretical studies of DNA origami melting were carried out in order to reveal the reversible association/disassociation process.

Folding of small origamis.

A model that preserves the known thermodynamic properties of double stranded DNA is introduced to study the formation of more complex DNA constructions, such as small origamis or Holliday junctions. We show that the thermodynamic behaviour of these complex DNA constructions is not only given by their sequence but also by their topology.