Cohesin organizes chromatin loops at DNA replication factories.

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication.

Direct non transcriptional role of NF-Y in DNA replication.

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication.

Replication Stress is an Actionable Genetic Vulnerability in Desmoplastic Small Round Cell Tumors.

Desmoplastic small round cell tumor (DSRCT) is an aggressive sarcoma subtype that is driven by the EWS-WT1 chimeric transcription factor. The prognosis for DSRCT is poor, and major advances in treating DSCRT have not occurred for over two decades. To identify effective therapeutic approaches to target DSRCT, we conducted a high-throughput drug sensitivity screen in a DSRCT cell line assessing chemosensitivity profiles for 79 small-molecule inhibitors. DSRCT cells were sensitive to PARP and ATR inhibitors (PARPi, ATRi), as monotherapies and in combination. These effects were recapitulated using multiple clinical PARPi and ATRi in three biologically distinct, clinically-relevant models of DSRCT, including cell lines, a patient-derived xenograft (PDX)-derived organoid model, and a cell line-derived xenograft mouse model. Mechanistically, exposure to a combination of PARPi and ATRi caused increased DNA damage, G2/M checkpoint activation, micronuclei accumulation, replication stress, and R-loop formation. EWS-WT1 silencing abrogated these phenotypes and was epistatic with exogenous expression of the R-loop resolution enzyme RNase H1 in reversing the sensitivity to PARPi and ATRi monotherapies. The combination of PARPi and ATRi also induced EWS-WT1-dependent cell-autonomous activation of the cGAS/STING innate immune pathway and cell surface expression of PD-L1. Taken together, these findings point towards a role for EWS-WT1 in generating R-loop-dependent replication stress that leads to a targetable vulnerability, providing a rationale for the clinical assessment of PARPi and ATRi in DSRCT.

Tipin/Tim1/And1 protein complex promotes Pol alpha chromatin binding and sister chromatid cohesion.

The Tipin/Tim1 complex plays an important role in the S-phase checkpoint and replication fork stability. However, the biochemical function of this complex is poorly understood. Using Xenopus laevis egg extract we show that Tipin is required for DNA replication in the presence of limiting amount of replication origins. Under these conditions the DNA replication defect correlates with decreased levels of DNA Polalpha on chromatin. We identified And1, a Polalpha chromatin-loading factor, as new Tipin-binding partner. We found that both Tipin and And1 promote stable binding of Polalpha to chromatin and that this is required for DNA replication under unchallenged conditions. Strikingly, extracts lacking Tipin and And1 also show reduced sister chromatids cohesion. These data indicate that Tipin/Tim1/And1 form a complex that links stabilization of replication fork and establishment of sister chromatid cohesion.

Essential global role of CDC14 in DNA synthesis revealed by chromosome underreplication unrecognized by checkpoints in cdc14 mutants.

The CDC14 family of multifunctional evolutionarily conserved phosphatases includes major regulators of mitosis in eukaryotes and of DNA damage response in humans. The CDC14 function is also crucial for accurate chromosome segregation, which is exemplified by its absolute requirement in yeast for the anaphase segregation of nucleolar organizers; however the nature of this essential pathway is not understood. Upon investigation of the rDNA nondisjunction phenomenon, it was found that cdc14 mutants fail to complete replication of this locus. Moreover, other late-replicating genomic regions (10% of the genome) are also underreplicated in cdc14 mutants undergoing anaphase. This selective genome-wide replication defect is due to dosage insufficiency of replication factors in the nucleus, which stems from two defects, both contingent on the reduced CDC14 function in the preceding mitosis. First, a constitutive nuclear import defect results in a drastic dosage decrease for those replication proteins that are regulated by nuclear transport. Particularly, essential RPA subunits display both lower mRNA and protein levels, as well as abnormal cytoplasmic localization. Second, the reduced transcription of MBF and SBF-controlled genes in G1 leads to the reduction in protein levels of many proteins involved in DNA replication. The failure to complete replication of late replicons is the primary reason for chromosome nondisjunction upon CDC14 dysfunction. As the genome-wide slow-down of DNA replication does not trigger checkpoints [Lengronne A, Schwob E (2002) Mol Cell 9:1067-1078], CDC14 mutations pose an overwhelming challenge to genome stability, both generating chromosome damage and undermining the checkpoint control mechanisms.

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae.

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome segregation. As DNA synthesis defects underlie chromosome rearrangements, monitoring DNA replication has become essential to understand the basis of genome instability. Saccharomyces cerevisiae is a classical model to study cell cycle regulation, but key DNA replication parameters, such as the fraction of cells in the S phase or the S-phase duration, are still difficult to determine. This protocol uses short and non-toxic pulses of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analog, in engineered TK-hENT1 yeast cells, followed by its detection by Click reaction to allow the visualization and quantification of DNA replication with high spatial and temporal resolution at both the single-cell and population levels by microscopy and flow cytometry. This method may identify previously overlooked defects in the S phase and cell cycle progression of yeast mutants, thereby allowing the characterization of new players essential for ensuring genome stability.

Measuring S-Phase Duration from Asynchronous Cells Using Dual EdU-BrdU Pulse-Chase Labeling Flow Cytometry.

Eukaryotes duplicate their chromosomes during the cell cycle S phase using thousands of initiation sites, tunable fork speed and megabase-long spatio-temporal replication programs. The duration of S phase is fairly constant within a given cell type, but remarkably plastic during development, cell differentiation or various stresses. Characterizing the dynamics of S phase is important as replication defects are associated with genome instability, cancer and ageing. Methods to measure S-phase duration are so far indirect, and rely on mathematical modelling or require cell synchronization. We describe here a simple and robust method to measure S-phase duration in cell cultures using a dual EdU-BrdU pulse-labeling regimen with incremental thymidine chases, and quantification by flow cytometry of cells entering and exiting S phase. Importantly, the method requires neither cell synchronization nor genome engineering, thus avoiding possible artifacts. It measures the duration of unperturbed S phases, but also the effect of drugs or mutations on it. We show that this method can be used for both adherent and suspension cells, cell lines and primary cells of different types from human, mouse and Drosophila. Interestingly, the method revealed that several commonly-used cancer cell lines have a longer S phase compared to untransformed cells.

Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication.

The six main minichromosome maintenance proteins (Mcm2-7), which presumably constitute the core of the replicative DNA helicase, are present in chromatin in large excess relative to the number of active replication forks. To evaluate the relevance of this apparent surplus of Mcm2-7 complexes in human cells, their levels were down-regulated by using RNA interference. Interestingly, cells continued to proliferate for several days after the acute (>90%) reduction of Mcm2-7 concentration. However, they became hypersensitive to DNA replication stress, accumulated DNA lesions, and eventually activated a checkpoint response that prevented mitotic division. When this checkpoint was abrogated by the addition of caffeine, cells quickly lost viability, and their karyotypes revealed striking chromosomal aberrations. Single-molecule analyses revealed that cells with a reduced concentration of Mcm2-7 complexes display normal fork progression but have lost the potential to activate "dormant" origins that serve a backup function during DNA replication. Our data show that the chromatin-bound "excess" Mcm2-7 complexes play an important role in maintaining genomic integrity under conditions of replicative stress.

Use of DNA combing for studying DNA replication in vivo in yeast and mammalian cells.

Plasticity is an inherent feature of chromosomal DNA replication in eukaryotes. Potential origins of DNA replication are made in excess, but are used (fired) in a partly stochastic, partly programmed manner throughout the S phase of the cell cycle. Since most origins have a firing efficiency below 50%, population-based analysis methods yield a cumulative picture of origin activity (obtained by accretion) that does not accurately describe how chromosomes are replicated in single cells. DNA combing is a method that allows the alignment on silanized glass coverslips, at high density and with uniform stretching, of single DNA molecules in the Mb range. If this DNA is isolated from cells that have been labelled with halogenated nucleotides (BrdU, CldU, IdU), it is possible to determine the density and position of replication origins as well as the rate and symmetry of fork progression, quantitatively and on single DNA molecules. This chapter will successively describe (a) the preparation ofsilanized coverslips, (b) the incorporation of halogenated nucleotides in newly synthesized DNA in yeast and mammalian cell lines, (c) the preparation and combing of genomic DNA, and finally (d) the acquisition and analysis of single-molecule images to extract salient features of replication dynamics.

Long-Term Imaging of DNA Damage and Cell Cycle Progression in Budding Yeast Using Spinning Disk Confocal Microscopy.

Live cell imaging can monitor biological processes in time and space by providing quantitative measurements of cell behavior on a single-cell basis and in live conditions. However the illumination required to visualize fluorescently tagged endogenous proteins often perturbs cellular physiology, a problem particularly acute for yeast cells that are small, highly photosensitive and with scarce protein content. Analyzing the activation of the DNA damage response (DDR) in various yeast mutants or growth conditions, as well as its consequences for cell cycle progression and cell viability over extended periods of time therefore requires a special microscopy setup that does not by itself create DNA damage or perturb cell growth. Here, we provide a quick guide, strains and advice for imaging the DDR in S. cerevisiae for extended time (3-12 h) using spinning-disk confocal microscopy in conditions of limited photobleaching and photodamage. DDR is a conserved mechanism that allows the cell to respond to various stresses, especially those altering DNA integrity or topology. Acquiring time-lapse images of the DDR at high temporal and spatial resolution is of great interest, in particular when studying the effects of mutations or drugs which compromise genomic stability and cell cycle progression.

Identification of Tah11/Sid2 as the ortholog of the replication licensing factor Cdt1 in Saccharomyces cerevisiae.

Faithful duplication of the genetic material requires that replication origins fire only once per cell cycle. Central to this control is the tightly regulated formation of prereplicative complexes (preRCs) at future origins of DNA replication. In all eukaryotes studied, this entails loading by Cdc6 of the Mcm2-7 helicase next to the origin recognition complex (ORC). More recently, another factor, named Cdt1, was shown to be essential for Mcm loading in fission yeast and Xenopus as well as for DNA replication in Drosophila and humans. Surprisingly, no Cdt1 homolog was found in budding yeast, despite the conserved nature of origin licensing. Here we identify Tah11/Sid2, previously isolated through interactions with topoisomerase and Cdk inhibitor mutants, as an ortholog of Cdt1. We show that sid2 mutants lose minichromosomes in an ARS number-dependent manner, consistent with ScCdt1/Sid2 being involved in origin licensing. Accordingly, cells partially depleted of Cdt1 replicate DNA from fewer origins, whereas fully depleted cells fail to load Mcm2 on chromatin and fail to initiate but not elongate DNA synthesis. We conclude that origin licensing depends in S. cerevisiae as in other eukaryotes on both Cdc6 and Cdt1.

Homeostatic control of START through negative feedback between Cln3-Cdk1 and Rim15/Greatwall kinase in budding yeast.

How cells coordinate growth and division is key for size homeostasis. Phosphorylation by G1-CDK of Whi5/Rb inhibitors of SBF/E2F transcription factors triggers irreversible S-phase entry in yeast and metazoans, but why this occurs at a given cell size is not fully understood. We show that the yeast Rim15-Igo1,2 pathway, orthologous to Gwl-Arpp19/ENSA, is up-regulated in early G1 and helps promoting START by preventing PP2ACdc55 to dephosphorylate Whi5. RIM15 overexpression lowers cell size while IGO1,2 deletion delays START in cells with low CDK activity. Deletion of WHI5, CDC55 and ectopic CLN2 expression suppress the START delay of igo1,2∆ cells. Rim15 activity increases after cells switch from fermentation to respiration, where Igo1,2 contribute to chromosome maintenance. Interestingly Cln3-Cdk1 also inhibits Rim15 activity, which enables homeostatic control of Whi5 phosphorylation and cell cycle entry. We propose that Rim15/Gwl regulation of PP2A plays a hitherto unappreciated role in cell size homeostasis during metabolic rewiring of the cell cycle.

Ensa controls S-phase length by modulating Treslin levels.

The Greatwall/Ensa/PP2A-B55 pathway is essential for controlling mitotic substrate phosphorylation and mitotic entry. Here, we investigate the effect of the knockdown of the Gwl substrate, Ensa, in human cells. Unexpectedly, Ensa knockdown promotes a dramatic extension of S phase associated with a lowered density of replication forks. Notably, Ensa depletion results in a decrease of Treslin levels, a pivotal protein for the firing of replication origins. Accordingly, the extended S phase in Ensa-depleted cells is completely rescued by the overexpression of Treslin. Our data herein reveal a new mechanism by which normal cells regulate S-phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.The Greatwall/Ensa/PP2A-B55 pathway controls mitotic substrate phosphorylation and mitotic entry. Here the authors show that cells regulate S phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.

Flexibility and governance in eukaryotic DNA replication.

Eukaryotic DNA replication begins at numerous but often poorly characterized sequences called origins, which are distributed fairly regularly along chromosomes. The elusive and idiosyncratic nature of origins in higher eukaryotes is now understood as resulting from a strong epigenetic influence on their specification, which provides flexibility in origin selection and allows for tailoring the dynamics of chromosome replication to the specific needs of cells. By contrast, the factors that assemble in trans to make these origins competent for replication and the kinases that trigger initiation are well conserved. Genome-wide and single-molecule approaches are being developed to elucidate the dynamics of chromosome replication. The notion that a well-coordinated progression of replication forks is crucial for many aspects of the chromosome cycle besides simply duplication begins to be appreciated.

Single-molecule analysis of DNA replication reveals novel features in the divergent eukaryotes Leishmania and Trypanosoma brucei versus mammalian cells.

Leishmania and Trypanosoma are unicellular parasites that possess markedly original biological features as compared to other eukaryotes. The Leishmania genome displays a constitutive 'mosaic aneuploidy', whereas in Trypanosoma brucei, the megabase-sized chromosomes are diploid. We accurately analysed DNA replication parameters in three Leishmania species and Trypanosoma brucei as well as mouse embryonic fibroblasts (MEF). Active replication origins were visualized at the single molecule level using DNA molecular combing. More than one active origin was found on most DNA fibres, showing that the chromosomes are replicated from multiple origins. Inter-origin distances (IODs) were measured and found very large in trypanosomatids: the mean IOD was 160 kb in T. brucei and 226 kb in L. mexicana. Moreover, the progression of replication forks was faster than in any other eukaryote analyzed so far (mean velocity 1.9 kb/min in T. brucei and 2.4-2.6 kb/min in Leishmania). The estimated total number of active DNA replication origins in trypanosomatids is ~170. Finally, 14.4% of unidirectional replication forks were observed in T. brucei, in contrast to 1.5-1.7% in Leishmania and 4% in MEF cells. The biological significance of these original features is discussed.

EdU Incorporation for FACS and Microscopy Analysis of DNA Replication in Budding Yeast.

DNA replication is a key determinant of chromosome segregation and stability in eukaryotes. The yeast Saccharomyces cerevisiae has been extensively used for cell cycle studies, yet simple but key parameters such as the fraction of cells in S phase in a population or the subnuclear localization of DNA synthesis have been difficult to gather for this organism. 5-ethynyl-2'-deoxyuridine (EdU) is a thymidine analogue that can be incorporated in vivo and later detected using copper-catalyzed azide alkyne cycloaddition (Click reaction) without prior DNA denaturation. This chapter describes a budding yeast strain and conditions that allow rapid EdU incorporation at moderate extracellular concentrations, followed by its efficient detection for the analysis of DNA replication in single cells by flow cytometry and fluorescence microscopy.

Analyzing the dynamics of DNA replication in Mammalian cells using DNA combing.

How cells duplicate their chromosomes is a key determinant of cell identity and genome stability. DNA replication can initiate from more than 100,000 sites distributed along mammalian chromosomes, yet a given cell uses only a subset of these origins due to inefficient origin activation and regulation by developmental or environmental cues. An impractical consequence of cell-to-cell variations in origin firing is that population-based techniques do not accurately describe how chromosomes are replicated in single cells. DNA combing is a biophysical DNA fiber stretching method which permits visualization of ongoing DNA synthesis along Mb-sized single-DNA molecules purified from cells that were previously pulse-labeled with thymidine analogues. This allows quantitative measurements of several salient features of chromosome replication dynamics, such as fork velocity, fork asymmetry, inter-origin distances, and global instant fork density. In this chapter we describe how to obtain this information from asynchronous cultures of mammalian cells.

DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity.

Deoxyribonucleic acid (DNA) replication and chromosome segregation must occur in ordered sequence to maintain genome integrity during cell proliferation. Checkpoint mechanisms delay mitosis when DNA is damaged or upon replication stress, but little is known on the coupling of S and M phases in unperturbed conditions. To address this issue, we postponed replication onset in budding yeast so that DNA synthesis is still underway when cells should enter mitosis. This delayed mitotic entry and progression by transient activation of the S phase, G2/M, and spindle assembly checkpoints. Disabling both Mec1/ATR- and Mad2-dependent controls caused lethality in cells with deferred S phase, accompanied by Rad52 foci and chromosome missegregation. Thus, in contrast to acute replication stress that triggers a sustained Mec1/ATR response, multiple pathways cooperate to restrain mitosis transiently when replication forks progress unhindered. We suggest that these surveillance mechanisms arose when both S and M phases were coincidently set into motion by a unique ancestral cyclin-Cdk1 complex.

Metabolic and environmental conditions determine nuclear genomic instability in budding yeast lacking mitochondrial DNA.

Mitochondrial dysfunctions are an internal cause of nuclear genome instability. Because mitochondria are key regulators of cellular metabolism, we have investigated a potential link between external growth conditions and nuclear chromosome instability in cells with mitochondrial defects. Using Saccharomyces cerevisiae, we found that cells lacking mitochondrial DNA (rho0 cells) have a unique feature, with nuclear chromosome instability that occurs in nondividing cells and strongly fluctuates depending on the cellular environment. Calorie restriction, lower growth temperatures, growth at alkaline pH, antioxidants (NAC, Tiron), or presence of nearby wild-type cells all efficiently stabilize nuclear genomes of rho0 cells, whereas high glucose and ethanol boost instability. In contrast, other respiratory mutants that still possess mitochondrial DNA (RHO(+)) keep fairly constant instability rates under the same growth conditions, like wild-type or other RHO(+) controls. Our data identify mitochondrial defects as an important driver of nuclear genome instability influenced by environmental factors.

A role for DNA polymerase θ in the timing of DNA replication.

Although DNA polymerase θ (Pol θ) is known to carry out translesion synthesis and has been implicated in DNA repair, its physiological function under normal growth conditions remains unclear. Here we present evidence that Pol θ plays a role in determining the timing of replication in human cells. We find that Pol θ binds to chromatin during early G1, interacts with the Orc2 and Orc4 components of the Origin recognition complex and that the association of Mcm proteins with chromatin is enhanced in G1 when Pol θ is downregulated. Pol θ-depleted cells exhibit a normal density of activated origins in S phase, but early-to-late and late-to-early shifts are observed at a number of replication domains. Pol θ overexpression, on the other hand, causes delayed replication. Our results therefore suggest that Pol θ functions during the earliest steps of DNA replication and influences the timing of replication initiation.