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 next-generation sequencing-chess problem.

The development of next-generation sequencing (NGS) technologies paved the way for studying the spatiotemporal coordination of cellular processes along the genome. However, data sets are commonly limited to a few time points, and missing information needs to be interpolated. Most models assume that the studied dynamics are similar between individual cells, so that a homogeneous cell culture can be represented by a population-wide average.

A genome-wide comprehensive analysis of nucleosome positioning in yeast.

In eukaryotic cells, the one-dimensional DNA molecules need to be tightly packaged into the spatially constraining nucleus. Folding is achieved on its lowest level by wrapping the DNA around nucleosomes. Their arrangement regulates other nuclear processes, such as transcription and DNA repair.

Rif1 restrains the rate of replication origin firing in Xenopus laevis.

Metazoan genomes are duplicated by the coordinated activation of clusters of replication origins at different times during S phase, but the underlying mechanisms of this temporal program remain unclear during early development. Rif1, a key replication timing factor, inhibits origin firing by recruiting protein phosphatase 1 (PP1) to chromatin counteracting S phase kinases. We have previously described that Rif1 depletion accelerates early Xenopus laevis embryonic cell cycles.

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.

Functional interplay between Mediator and RSC chromatin remodeling complex controls nucleosome-depleted region maintenance at promoters.

Chromatin organization is crucial for transcriptional regulation in eukaryotes. Mediator is an essential and conserved co-activator thought to act in concert with chromatin regulators. However, it remains largely unknown how their functions are coordinated.

A quantitative modelling approach for DNA repair on a population scale.

The great advances of sequencing technologies allow the in vivo measurement of nuclear processes-such as DNA repair after UV exposure-over entire cell populations. However, data sets usually contain only a few samples over several hours, missing possibly important information in between time points. We developed a data-driven approach to analyse CPD repair kinetics over time in Saccharomyces cerevisiae.

Genomic analysis of Rad26 and Rad1-Rad10 reveals differences in their dependence on Mediator and RNA polymerase II.

Mediator is a conserved coregulator playing a key role in RNA polymerase (Pol) II transcription. Mediator also links transcription and nucleotide excision repair (NER) via a direct contact with Rad2/ERCC5(XPG) endonuclease. In this work, we analyzed the genome-wide distribution of Rad26/ERCC6(CSB) and Rad1-Rad10/ERCC4(XPF)-ERCC1, addressing the question of a potential link of these proteins with Mediator and Pol II in yeast Our genomic analyses reveal that Rad1-Rad10 and Rad26 are present on the yeast genome in the absence of genotoxic stress, especially at highly transcribed regions, with Rad26 binding strongly correlating with that of Pol II.

Polo-like kinase 1 (Plk1) regulates DNA replication origin firing and interacts with Rif1 in Xenopus.

The activation of eukaryotic DNA replication origins needs to be strictly controlled at multiple steps in order to faithfully duplicate the genome and to maintain its stability. How the checkpoint recovery and adaptation protein Polo-like kinase 1 (Plk1) regulates the firing of replication origins during non-challenged S phase remained an open question. Using DNA fiber analysis, we show that immunodepletion of Plk1 in the Xenopus in vitro system decreases replication fork density and initiation frequency.

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.

Polo-like kinase 1 (Plk1) is a positive regulator of DNA replication in the system.

Polo-like kinase 1 (Plk1) is a cell cycle kinase essential for mitosis progression, but also important for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is expressed in S phase, little is known about its function during unperturbed DNA replication. Using egg extracts, mimicking early embryonic replication, we demonstrate that Plk1 is simultaneously recruited to chromatin with pre-replication proteins where it accumulates throughout S phase.

On the Interplay of the DNA Replication Program and the Intra-S Phase Checkpoint Pathway.

DNA replication in eukaryotes is achieved by the activation of multiple replication origins which needs to be precisely coordinated in space and time. This spatio-temporal replication program is regulated by many factors to maintain genome stability, which is frequently threatened through stresses of exogenous or endogenous origin. Intra-S phase checkpoints monitor the integrity of DNA synthesis and are activated when replication forks are stalled.

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.

Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations.

We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations, and by taking into account the chromatin's fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement with corresponding experimental data from both S.

Tight Chk1 Levels Control Replication Cluster Activation in Xenopus.

DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal program. The ATR/Chk1 dependent replication checkpoint inhibits the activation of later firing origins. In the Xenopus in vitro system initiations are not sequence dependent and 2-5 origins are grouped in clusters that fire at different times despite a very short S phase.

From simple bacterial and archaeal replicons to replication N/U-domains.

The Replicon Theory proposed 50 years ago has proven to apply for replicons of the three domains of life. Here, we review our knowledge of genome organization into single and multiple replicons in bacteria, archaea and eukarya. Bacterial and archaeal replicator/initiator systems are quite specific and efficient, whereas eukaryotic replicons show degenerate specificity and efficiency, allowing for complex regulation of origin firing time.

Multiscale analysis of genome-wide replication timing profiles using a wavelet-based signal-processing algorithm.

In this protocol, we describe the use of the LastWave open-source signal-processing command language (http://perso.ens-lyon.fr/benjamin.

Replication fork polarity gradients revealed by megabase-sized U-shaped replication timing domains in human cell lines.

In higher eukaryotes, replication program specification in different cell types remains to be fully understood. We show for seven human cell lines that about half of the genome is divided in domains that display a characteristic U-shaped replication timing profile with early initiation zones at borders and late replication at centers. Significant overlap is observed between U-domains of different cell lines and also with germline replication domains exhibiting a N-shaped nucleotide compositional skew.

Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome.

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.

Do replication forks control late origin firing in Saccharomyces cerevisiae?

Recent studies of eukaryotic DNA replication timing profiles suggest that the time-dependent rate of origin firing, I(t), has a universal shape, which ensures a reproducible replication completion time. However, measurements of I(t) are based on population averages, which may bias the shape of the I(t) because of imperfect cell synchrony and cell-to-cell variability. Here, we measure the population-averaged I(t) profile from synchronized Saccharomyces cerevisiae cells using DNA combing and we extract the single-cell I(t) profile using numerical deconvolution.

DNA replication induces compositional biases in yeast.

Asymmetries intrinsic to the process of DNA replication are expected to cause differences in the substitution patterns of the leading and the lagging strands and to induce compositional biases. These biases have been detected in the majority of eubacterial genomes but rarely in eukaryotes. Only in the human genome, the activity of a minority of replication origins seems to generate compositional biases.

Giant yeast cells with nonrecyclable ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides and thereby provides the precursors required for DNA synthesis and repair. In an attempt to test cell resistance to a permanent replicational stress, we constructed a mutant Saccharomyces cerevisiae strain containing exclusively nonrecyclable catalytic subunits of RNR that become inactivated following the reduction of one ribonucleoside diphosphate. In this rnr1C883A rnr3Δ mutant, the synthesis of each deoxyribonucleotide thus requires the production of one Rnr1C883A protein, which means that 26 million Rnr1C883A proteins (half the protein complement of a wild-type cell) have to be produced during each cell cycle.

Mathematical modelling of eukaryotic DNA replication.

Eukaryotic DNA replication is a complex process. Replication starts at thousand origins that are activated at different times in S phase and terminates when converging replication forks meet. Potential origins are much more abundant than actually fire within a given S phase.

Use of DNA combing to study DNA replication in Xenopus and human cell-free systems.

The Xenopus egg extract has become the gold standard for in vitro studies of metazoan DNA replication. We have used this system to study the mechanisms that ensure rapid and complete DNA replication despite random initiation during Xenopus early development. To this end we adapted the DNA combing technique to investigate the distribution of replication bubbles along single DNA molecules.

Universal temporal profile of replication origin activation in eukaryotes.

Although replication proteins are conserved among eukaryotes, the sequence requirements for replication initiation differ between species. In all species, however, replication origins fire asynchronously throughout S phase. The temporal program of origin firing is reproducible in cell populations but largely probabilistic at the single-cell level.