The organization of genome duplication is a critical determinant of the landscape of genome maintenance.

Genome duplication is essential for cell proliferation, and the mechanisms regulating its execution are highly conserved. These processes give rise to a spatiotemporal organization of replication initiation across the genome, referred to as the replication program. Despite the identification of such programs in diverse eukaryotic organisms, their biological importance for cellular physiology remains largely unexplored. We address this fundamental question in the context of genome maintenance, taking advantage of the inappropriate origin firing that occurs when fission yeast cells lacking the Rad3/ATR checkpoint kinase are subjected to replication stress. Using this model, we demonstrate that the replication program quantitatively dictates the extent of origin de-regulation and the clustered localization of these events. Furthermore, our results uncover an accumulation of abnormal levels of single-stranded DNA (ssDNA) and the Rad52 repair protein at de-regulated origins. We show that these loci constitute a defining source of the overall ssDNA and Rad52 hotspots in the genome, generating a signature pattern of instability along the chromosomes. We then induce a genome-wide reprogramming of origin usage and evaluate its consequences in our experimental system. This leads to a complete redistribution of the sites of both inappropriate initiation and associated Rad52 recruitment. We therefore conclude that the organization of genome duplication governs the checkpoint control of origin-associated hotspots of instability and plays an integral role in shaping the landscape of genome maintenance.

CDK activity provides temporal and quantitative cues for organizing genome duplication.

In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection. We have addressed this question in the fission yeast, taking advantage of a simplified cell cycle network in which cell proliferation is driven by a single cyclin-CDK module. This system allows us to precisely control CDK activity in vivo using chemical genetics. First, in contrast to previous reports, our results clearly show that distinct cyclin-CDK pairs are not essential for regulating specific subsets of origins and for establishing a normal replication program. Importantly, we then demonstrate that the timing at which CDK activity reaches the S phase threshold is critical for the organization of replication in distinct efficiency domains, while the level of CDK activity at the onset of S phase is a dose-dependent modulator of overall origin efficiencies. Our study therefore implicates these different aspects of CDK regulation as versatile mechanisms for shaping the architecture of DNA replication across the genome.

Thermoplastic elastomer with advanced hydrophilization and bonding performances for rapid (30 s) and easy molding of microfluidic devices.

One of the most important areas of research on microfluidic technologies focuses on the identification and characterisation of novel materials with enhanced properties and versatility. Here we present a fast, easy and inexpensive microstructuration method for the fabrication of novel, flexible, transparent and biocompatible microfluidic devices. Using a simple hot press, we demonstrate the rapid (30 s) production of various microfluidic prototypes embossed in a commercially available soft thermoplastic elastomer (sTPE). This styrenic block copolymer (BCP) material is as flexible as PDMS and as thermoformable as classical thermoplastics. It exhibits high fidelity of replication using SU-8 and epoxy master molds in a highly convenient low-isobar (0.4 bar) and iso-thermal process. Microfluidic devices can then be easily sealed using either a simple hot plate or even a room-temperature assembly, allowing them to sustain liquid pressures of 2 and 0.6 bar, respectively. The excellent sorption and biocompatibility properties of the microchips were validated via a standard rhodamine dye assay as well as a sensitive yeast cell-based assay. The morphology and composition of the surface area after plasma treatment for hydrophilization purposes are stable and show constant and homogenous distribution of block nanodomains (∼22° after 4 days). These domains, which are evenly distributed on the nanoscale, therefore account for the uniform and convenient surface of a "microfluidic scale device". To our knowledge, this is the first thermoplastic elastomer material that can be used for fast and reliable fabrication and assembly of microdevices while maintaining a high and stable hydrophilicity.

Roles of CDK and DDK in Genome Duplication and Maintenance: Meiotic Singularities.

Cells reproduce using two types of divisions: mitosis, which generates two daughter cells each with the same genomic content as the mother cell, and meiosis, which reduces the number of chromosomes of the parent cell by half and gives rise to four gametes. The mechanisms that promote the proper progression of the mitotic and meiotic cycles are highly conserved and controlled. They require the activities of two types of serine-threonine kinases, the cyclin-dependent kinases (CDKs) and the Dbf4-dependent kinase (DDK). CDK and DDK are essential for genome duplication and maintenance in both mitotic and meiotic divisions. In this review, we aim to highlight how these kinases cooperate to orchestrate diverse processes during cellular reproduction, focusing on meiosis-specific adaptions of their regulation and functions in DNA metabolism.

A drug-compatible and temperature-controlled microfluidic device for live-cell imaging.

Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging.

The programme of DNA replication: beyond genome duplication.

The accurate duplication and transmission of genetic information is critical for cell growth and proliferation, and this is ensured in part by the multi-layered regulation of DNA synthesis. One of the key steps in this process is the selection and activation of the sites of replication initiation, or origins, across the genome. Interestingly, origin usage changes during development and in different pathologies, suggesting an integral interplay between the establishment of replication initiation along the chromosomes and cellular function. The present review discusses how the spatiotemporal organization of replication origin activation may play crucial roles in the control of biological events.

The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor.

In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.

G1/S transcription and the DNA synthesis checkpoint: common regulatory mechanisms.

When DNA replication is challenged, cells activate a DNA-synthesis checkpoint blocking cell cycle progression until they are able to overcome the replication defects. In fission yeast, Cds1 is the effector kinase of this checkpoint, inhibiting M phase entry through inactivation of the phosphatase Cdc25, stabilizing stalled replication forks to prevent deleterious DNA structures and triggering transcriptional activation of S-phase genes. The MBF complex controls the transcription of genes required for the S phase and Yox1, a homeodomain-containing protein, binds and represses MBF-dependent transcription at the end of S phase in a cell cycle-regulated manner. Interestingly, when the DNA synthesis checkpoint is activated, Yox1 is phosphorylated by Cds1 resulting in the abrogation of its binding to MBF. As a consequence, MBF-dependent transcription is maintained active until cells are able to overcome the replication challenge. Thus, Yox1 couples normal cell cycle regulation and the DNA synthesis checkpoint in a single transcriptional complex.

Yox1 links MBF-dependent transcription to completion of DNA synthesis.

When DNA replication is challenged cells activate a DNA synthesis checkpoint, blocking cell cycle progression until they are able to overcome the replication defects. In fission yeast, Cds1 is the effector kinase of this checkpoint, inhibiting M-phase entry, stabilizing stalled replication forks and triggering transcriptional activation of S-phase genes. The molecular basis of this last effect is largely unknown. The Mlu1 binding factor (MBF) complex controls the transcription of S-phase genes. We purified novel interactors of the MBF complex and identified the repressor Yox1. When the DNA synthesis checkpoint is activated, Yox1 is phosphorylated, which abrogates its binding to MBF. MBF-dependent transcription therefore remains active until cells are able to overcome this challenge.

Promoter-driven splicing regulation in fission yeast.

The meiotic cell cycle is modified from the mitotic cell cycle by having a pre-meiotic S phase that leads to high levels of recombination, two rounds of nuclear division with no intervening DNA synthesis and a reductional pattern of chromosome segregation. Rem1 is a cyclin that is only expressed during meiosis in the fission yeast Schizosaccharomyces pombe. Cells in which rem1 has been deleted show decreased intragenic meiotic recombination and a delay at the onset of meiosis I (ref. 1). When ectopically expressed in mitotically growing cells, Rem1 induces a G1 arrest followed by severe mitotic catastrophes. Here we show that rem1 expression is regulated at the level of both transcription and splicing, encoding two proteins with different functions depending on the intron retention. We have determined that the regulation of rem1 splicing is not dependent on any transcribed region of the gene. Furthermore, when the rem1 promoter is fused to other intron-containing genes, the chimaeras show a meiotic-specific regulation of splicing, exactly the same as endogenous rem1. This regulation is dependent on two transcription factors of the forkhead family, Mei4 (ref. 2) and Fkh2 (ref. 3). Whereas Mei4 induces both transcription and splicing of rem1, Fkh2 is responsible for the intron retention of the transcript during vegetative growth and the pre-meiotic S phase.