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The computational design of synthetic DNA sequences with desired in vivo properties is gaining traction in the field of synthetic genomics. We propose here a computational method which combines a kinetic Monte Carlo framework with a deep mutational screening based on deep learning predictions. We apply our method to build regular nucleosome arrays with tailored nucleosomal repeat lengths (NRL) in yeast. Our design is validated in vivo by successfully engineering and integrating thousands of kilobases long tandem arrays of computationally optimized sequences which could accommodate NRLs much larger than the yeast natural NRL. This method delineates the key sequence rules for nucleosome positioning in yeast and is readily applicable to other sequence properties and other genomes.

G-rich sequences found at multiple sites throughout all genomes may form secondary structures called G-quadruplexes (G4), which act as roadblocks for molecular motors. Among the enzymes thought to process these structures, the Pif1 DNA helicase is considered as an archetypical G4-resolvase and its absence has been linked to G4-related genomic instabilities in yeast. Here we developed a single-molecule assay to observe Pif1 opening a DNA duplex and resolving the G4 in real time. In support of former enzymological studies, we show that the helicase reduces the lifetime of G4 from hours to seconds. However, we observe that in the presence of a G4, Pif1 exhibits a strong strand switching behavior, which can lead to Pif1 escaping G4 resolution, depending on the structural context surrounding the substrate. This behavior is also detected in the presence of other roadblocks (LNA or RNA). We propose that the efficiency of Pif1 to remove a roadblock (G4 or other) is affected by its strand switching behavior and depends on the context surrounding the obstacle. We discuss how this switching behavior may explain several aspects of Pif1 substrate preference and affect its activity as a G4 resolvase in vivo.

Milankovic cycles describe the changes in the Earth's orbit and rotation axis and their impact on its climate over thousands of years. Singular Spectrum Analysis (SSA) is a signal processing method that is best known for its ability to find and extract pseudo-cycles in complex signals. In this short paper, we propose to apply it to three time series that have been proposed as geological reference time scales, in order to retrieve, compare and identify their Milankovic periodicities: (1) LR04, a stack of Plio-Pleistocene benthic microfossil records (Lisiecki and Raymo, 2005), (2) the CO2 and CH4 records from the Vostok ice core (Petit et al, 1999) and (3) the long-term orbital solution La04 for the insolation of Laskar et al (2004). The Vostok CO2 and CH4 series share the first 7 SSA components, three main ones (98, 104, 39 kyr), and four smaller ones (18, 22, 65, 180 kyr). CO2 displays a component at 28 kyr and a doublet at 61 and 62 kyr. CH4 displays a doublet near 50 kyr. 18/22 ky is a precession doublet, 62 kyr an insolation component, and 95/105 kyr an insolation/eccentricity doublet. The 49/50 kyr doublet in CH4 is not found in the orbital model. The SSA results for the La04 orbital solution are in excellent agreement with the values obtained by Laskar et al (2004). Four SSA components of obliquity are almost identical (rounded figures are 41, 54, 29 and 39 kyr). As far as eccentricity is concerned, the first five components are 404, 95, 124, 99, and 132 kyr. The next components are not found in our list of components for eccentricity, but they are in the SSA of insolation, at 2338, 970, 488 and 684 kyr. With more than 20 components, the LR04 stack is the richest series. In order of decreasing amplitude, one encounters 41, 95 and 75 kyr components. Next are smaller 39.5 and 53.6 kyr components, and a 22.4 kyr component. One recognizes one of the two main precession components, the doublet of obliquity components, a line at 47.4 kyr that is not found in any of the other spectra, and a doublet at 53.6 and 55.7 kyr, corresponding to the line at 54 kyr found in all four orbital quantities. Next comes a line at 63.6 kyr that may correspond to a line in insolation, CH4 and CO2. Then come components from eccentricity variations at 75.2, 94.5, 107.2, 132.1, 198.6 and 400.9 kyr. The remaining components of LR04 show up in La04. The “elusive ~200 kyr eccentricity cycle” of Hilgen et al (2020) is actually present in all three series, in the La04 orbital model as a 195 ± 6 kyr component of eccentricity and in LR04 as a 198.6 ± 5.6 kyr component. Finding not only the main expected Milankovic periodicities but also many “secondary” components with much smaller amplitudes gives confidence in our iterative SSA method (iSSA), on the quality of the La04 model and on the remarkable LR04 sedimentary stack, with more than 15 “ Milankovic periods”.

International audience; The quest for small molecules that strongly bind to Gquadruplex-DNA (G4), so-called G4 ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to discover or rationally design G4 ligands, evaluate their G4interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and use them as innovative chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts aimed at developing small molecules that destabilize G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application in neurobiology as many genetic and age-related diseases are caused by an overrepresentation of G4s. Herein, we report on our efforts to develop in vitro assays to reliably identify molecules able to destabilize G4s. This workflow comprises the newly designed G4-unfold assay, adapted from the G4-helicase assay implemented with Pif1, as well as a series of biophysical and biochemical techniques classically used to study G4/ligand interactions (CD, UV−vis, PAGE, and FRET-melting), and a qPCR stop assay, adapted from a Taq-based protocol recently used to identify G4s in the genomic DNA of Schizosaccharomyces pombe. This unique, multipronged approach leads to the characterization of a phenylpyrrolocytosine (PhpC)-based G-clamp analog as a prototype of G4disrupting small molecule whose properties are validated through many different and complementary in vitro evaluations.

G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA replication, transcription or repair. While manyin-vitrostudies have focused on the kinetics of G4 formation within DNA single-strands, G4 are foundin-vivoin double-stranded DNA regions, where their formation is challenged by pairing between the two complementary strands. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on the persistence of G4in vivo. To address this issue, we designed a single molecule assay allowing measuring G4 folding and persistence while the structure is periodically challenged by the complementary strand. We quantified both the folding rate and the persistence time of biologically relevant G4 structures and showed that the dynamics of G4 formation depends strongly on the genomic location. G4 are found much more stable in promoter regions and replication origins than in telomeric regions. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence increased. Our assay opens new perspectives for the measurement of G4 dynamics, which is critical to understand their role in genetic regulation.

Fermented foods usually have long storage lives and interesting nutritional values, which makes fermentation a very popular transformation technique all over the world, including for drink production. Even without sound scientific proof, consumers are generally encouraged by popular media to improve their health through fermented diets that bring a good amount of useful probiotics, making kefir increasingly fashionable. Milk kefir, thought to have its origins in the north Caucasus mountains, is still consumed by a lot of Russians. It is traditionally made by inoculating milk with kefir grains and keeping it in skin bags at room temperature for a day or more, depending on the conditions and fermentation level desired. Biochemical, metagenomic and metabolomic studies provide evidence of the large number of microorganisms in starting strains and the variety of possible bioactive compounds that could be formed during fermentation, and have to be followed by medical/clinical studies aimed at characterizing the potential health effects of regular kefir consumption.

The quest for small molecules that avidly bind to G-quadruplex-DNA (G4-DNA, or G4), so called G4-ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to i- screen or design G4-ligands, ii- evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and iii- use them as unique chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts at developing small molecules aimed at destabilizing G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application to neurobiology as many genetic and age-related diseases are caused by an over-representation of G4s, itself caused by a deficiency of G4-resolving enzymes, the G4-helicases. Herein, we report on our double effort to i- develop a reliable in vitro assay to identify molecules able to destabilize G4s, the G4-unfold assay, and ii- fully characterize the first prototype of G4-disrupting small molecule, a phenylpyrrolcytosine (PhpC)-based G-clamp analog.

The so-called 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, in vivo application of this property has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro as expected, analysis of nucleosome occupancy on these arrays in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

Understanding the effect of an ever-growing number of human variants detected by genome sequencing is a medical challenge. The yeast Saccharomyces cerevisiae model has held attention for its capacity to monitor the functional impact of missense mutations found in human genes, including the BRCA1 breast and ovarian cancer susceptibility gene. When expressed in yeast, the wild-type full-length BRCA1 protein forms a single nuclear aggregate and induces a growth inhibition. Both events are modified by pathogenic mutations of BRCA1. However, the biological processes behind these events in yeast remain to be determined. Here, we show that the BRCA1 nuclear aggregation and the growth inhibition are sensitive to misfolding effects induced by missense mutations. Moreover, misfolding mutations impair the nuclear targeting of BRCA1 in yeast cells and in a human cell line. In conclusion, we establish a connection between misfolding and nuclear transport impairment, and we illustrate that yeast is a suitable model to decipher the effect of misfolding mutations.

International audience; Guanine-rich DNA strands can fold in vitro into non-canonical DNA structures called G-quadruplexes. These structures may be very stable under physiological conditions. Evidence suggests that G-quadruplex structures may act as 'knots' within ge-nomic DNA, and it has been hypothesized that proteins may have evolved to remove these structures. The first indication of how G-quadruplex structures could be unfolded enzymatically came in the late 1990s with reports that some well-known duplex DNA helicases resolved these structures in vitro. Since then, the number of studies reporting G-quadruplex DNA unfolding by helicase enzymes has rapidly increased. The present review aims to present a general overview of the helicase/G-quadruplex field.

Pif1p is the prototypical member of the PIF1 family of DNA helicases, a subfamily of SFI helicases conserved from yeast to humans. Baker's yeast Pif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and may also have a general role in chromosomal replication by affecting Okazaki fragment maturation. Here we investigate the substrate preferences for Pif1p. The enzyme was preferentially active on RNA-DNA hybrids, as seen by faster unwinding rates on RNA-DNA hybrids compared to DNA-DNA hybrids. When using forked substrates, which have been shown previously to stimulate the enzyme, Pif1p demonstrated a preference for RNA-DNA hybrids. This preferential unwinding could not be correlated to preferential binding of Pif1p to the substrates that were the most readily unwound. Although the addition of the single-strand DNA-binding protein replication protein A (RPA) stimulated the helicase reaction on all substrates, it did not diminish the preference of Pif1p for RNA-DNA substrates. Thus, forked RNA-DNA substrates are the favored substrates for Pif1p in vitro. We discuss these findings in terms of the known biological roles of the enzyme.

The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals.

In eukaryotes, the genome is packed into chromosomes, each consisting of large polymeric fibers made of DNA bound with proteins (mainly histones) and RNA molecules. The nature and precise 3D organization of this fiber has been a matter of intense speculations and debates. In the emerging picture, the local chromatin state plays a critical role in all fundamental DNA transactions, such as transcriptional control, DNA replication or repair. However, the molecular and structural mechanisms involved remain elusive. The purpose of this review is to give an overview of the tremendous efforts that have been made for almost 40 years to build physiologically relevant models of chromatin structure. The motivation behind building such models was to shift our representation and understanding of DNA transactions from a too simplistic 'naked DNA' view to a more realistic 'coated DNA' view, as a step towards a better framework in which to interpret mechanistically the control of genetic expression and other DNA metabolic processes. The field has evolved from a speculative point of view towards in vitro biochemistry and in silico modeling, but is still longing for experimental in vivo validations of the proposed structures or even proof of concept experiments demonstrating a clear role of a given structure in a metabolic transaction. The mere existence of a chromatin fiber as a relevant biological entity in vivo has been put into serious questioning. Current research is suggesting a possible reconciliation between theoretical studies and experiments, pointing towards a view where the polymorphic and dynamic nature of the chromatin fiber is essential to support its function in genome metabolism.

Terminal deoxynucleotidyltransferase (TdT) catalyzes the addition of nucleotides to 3′-hydroxyl ends of DNA strands in a template-independent manner and has been shown to add N-regions to gene segment junctions during V(D)J recombination. TdT is highly conserved in all vertebrate species, with a second isoform, characterized by a 20-amino acid insertion near the COOH-terminal end, described only in the mouse. The two murine isoforms differ in their subcellular localization, and the long isoform (TdTL) has previously been found to be unable to add N-regions. Using purified protein produced in a high level expression system in Escherichia coli, we were able to carry out detailed catalytic comparisons of these two TdT isoforms. We discovered that TdTL exhibits terminal transferase activity with kinetic parameters similar to those of the conserved TdT isoform (TdTS). We observed, however, that TdTL is inactivated at physiologic temperature but stable at lower temperatures. This thermal sensitivity of TdTL polymerase activity is not correlated with a significant change in the circular dichroism spectrum of the protein. Thus, the 20-amino acid insertion in TdTL does not affect the catalytic activity but modifies the thermosensitivity.

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5'-triphosphates onto the 3'-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production in Escherichia coli of murine TdT with minimal proteolysis and high specific activity.

Genomes contain tandem repeats that are at risk of internal rearrangements and a threat to genome integrity. Here, we investigated the behavior of the human subtelomeric minisatellites HRAS1, CEB1, and CEB25 in Saccharomyces cerevisiae. In mitotically growing wild-type cells, these GC–rich tandem arrays stimulate the rate of gross chromosomal rearrangements (GCR) by 20, 1,620, and 276,000-fold, respectively. In the absence of the Pif1 helicase, known to inhibit GCR by telomere addition and to unwind G-quadruplexes, the GCR rate is further increased in the presence of CEB1, by 385-fold compared to the pif1Δ control strain. The behavior of CEB1 is strongly dependent on its capacity to form G-quadruplexes, since the treatment of WT cells with the Phen-DC3 G-quadruplex ligand has a 52-fold stimulating effect while the mutation of the G-quadruplex-forming motif reduced the GCR rate 30-fold in WT and 100-fold in pif1Δ cells. The GCR events are telomere additions within CEB1. Differently, the extreme stimulation of CEB25 GCR depends on its affinity for Cdc13, which binds the TG-rich ssDNA telomere overhang. This property confers a biased orientation-dependent behavior to CEB25, while CEB1 and HRAS1 increase GCR similarly in either orientation. Furthermore, we analyzed the minisatellites‚ distribution in the human genome and discuss their potential role to trigger subtelomeric rearrangements., Author Summary All genomes contain particular DNA sequences that are prone to break and rearrange. They can be lost or rescued at the expense of sequence variations and complex rearrangements. Using a sensitive yeast model system, we examined the fragility of the HRAS1, CEB1, and CEB25 GC-rich human minisatellites (tandem repetition of motifs from 10 to 100 bp long). We observed that they all stimulate Gross Chromosomal Rearrangements but to very different extents, both in wild type and in cells deficient for the Pif1 helicase. Several intrinsic sequence features can account for these differences: the total number of repeats, the ability to form G-quadruplex secondary structures, or the ability to bind with high affinity the telomerase cofactor Cdc13. The orientation on the chromosome dictates the type of GCR (telomere addition versus other structural rearrangements) while not affecting the GCR rate in most cases. Being enriched in the subtelomeric regions of the human chromosomes, this class of GC–rich minisatellite has the potential to trigger a large variety of human genome rearrangements.

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences., Author Summary Changes in the primary DNA sequence are a major source of pathologies and cancers. The hereditary information also resides in secondary DNA structures, a layer of genetic information that remains poorly understood. Biophysical and structural studies have long established that, in vitro, the DNA molecule can adopt diverse structures different from the canonical Watson-Crick conformations. However, for a long time their existence in vivo has been regarded with a certain skepticism and their functional role elusive. One example is the G-quadruplex structure, which involves G-quartets that form between four DNA strands. Here, using in vitro and in vivo assays in the yeast S. cerevisiae, we reveal the unexpected role of the Pif1 helicase in maintaining the stability of the human CEB1 G-rich tandem repeat array. By site-directed mutagenesis, we show that the genomic instability of CEB1 repeats in absence of Pif1 and is directly dependent on the ability of CEB1 to form G-quadruplex structures. We show that Pif1 is very efficient in vitro in processing G-quadruplex structures formed by CEB1. We propose that Pif1 maintains CEB1 repeats by its ability to resolve G-quadruplex structures, thus providing circumstantial evidence of their formation in vivo.

Improving NGS is the major challenge of the third-generation sequencing which is mainly focus on single molecule techniques. Single molecule offers the ultimate sensitivity, avoiding the amplification steps which introduces bias and strongly impairs epigenetic analysis. However, single molecule approaches requires minute signal detection which results in an increased error rate. We present here a third method where a single molecule can be interrogated in a repeated manner allowing good signal averaging and leading to very low error rate. This method may be seen as revisiting Sequencing By Hybridization (SBH) using a hairpin re-zipping blocking assay. In our single molecule micromanipulation assay we detect the hybridization of the oligonucleotides k-mers and their actual position along the target with an accuracy of a few bases. Each repeat is thus identified by its actual position. This process is repeated until all hybridization positions are known accurately. Assembling the target sequence is achieved after testing a series of oligonucleotides once the target sequence is sufficiently covered. The overlapping of the k-mers is used by a software to reconstruct the sequence.

The Pif1p family of DNA helicases is conserved from yeast to humans. To date, four members of this family have been analyzed in some detail by in vitro and in vivo assays: the two baker’s yeast helicases, ScPif1p and Rrm3p, the fission yeast Pfh1p and the human enzyme hPif1p. In vitro, these enzymes are 5 0 to 3 0 DNA helicase and show little processivity. In vivo, ScPif1p, Rrm3p and probably Pfh1p, function in both the nucleus at specific genomic loci and in mitochondria, where they are needed for the stable maintenance of the genome as accessory helicases to the replication machinery. Interestingly, they act on common DNA substrates but appear to have largely non-overlapping cellular functions, ranging from Okazaki fragment processing, telomerase inhibition, to helping the replication fork progress through non-nucleosomal protein–DNA complexes. For example, both ScPif1p and Rrm3p affect the replication of telomeres, but in a different way: Pif1p inhibits telomerase-mediated telomere elongation by directly removing telomerase from a DNA end, whereas Rrm3p facilitates replication through telomeric DNA. Here we review the current knowledge on the Pif1-like helicases, as a first step towards understanding the basis of their functional specialization and mechanism of action.

Telomeres are the physical ends of eukaryotic chromosomes. Genetic studies have established that the baker's yeast Pif1p DNA helicase is a negative regulator of telomerase, the specialized reverse transcriptase that maintains telomeric DNA, but the biochemical basis for this inhibition was unknown. Here we show that in vitro, Pif1p reduces the processivity of telomerase and releases telomerase from telomeric oligonucleotides. The released telomerase is enzymatically active because it is able to lengthen a challenger oligonucleotide. In vivo, overexpression of Pif1p reduces telomerase association with telomeres, whereas depleting cells of Pif1p increases the levels of telomere-bound Est1p, a telomerase subunit that is present on the telomere when telomerase is active. We propose that Pif1p helicase activity limits telomerase action both in vivo and in vitro by displacing active telomerase from DNA ends.

International audience; The crystal structure of the catalytic core of murine terminal deoxynucleotidyltransferase (TdT) at 2.35 A resolution reveals a typical DNA polymerase beta-like fold locked in a closed form. In addition, the structures of two different binary complexes, one with an oligonucleotide primer and the other with an incoming ddATP-Co(2+) complex, show that the substrates and the two divalent ions in the catalytic site are positioned in TdT in a manner similar to that described for the human DNA polymerase beta ternary complex, suggesting a common two metal ions mechanism of nucleotidyl transfer in these two proteins. The inability of TdT to accommodate a template strand can be explained by steric hindrance at the catalytic site caused by a long lariat-like loop, which is absent in DNA polymerase beta. However, displacement of this discriminating loop would be sufficient to unmask a number of evolutionarily conserved residues, which could then interact with a template DNA strand. The present structure can be used to model the recently discovered human polymerase mu, with which it shares 43% sequence identity.

Terminal deoxynucleotidyl transferase (TdT) catalyzes the condensation of deoxyribonucleotides on 3′-hydroxyl ends of DNA strands in a template-independent manner and adds N-regions to gene segment junctions during V(D)J recombination. Although TdT is able to incorporate a few ribonucleotides in vitro, TdT discrimination between ribo- and deoxyribonucleotides has never been studied. We found that TdT shows only a minor preference for incorporation of deoxyribonucleotides over ribonucleotides on DNA strands. However, incorporation of ribonucleotides alone or in the presence of deoxyribonucleotides generally leads to premature chain termination, reflecting an impeded accommodation of ribo- or mixed ribo/deoxyribonucleic acid substrates by TdT. An essential catalytic aspartate in TdT was identified, which is a first step toward understanding the apparent lack of sugar discrimination by TdT.

A large number of missense mutations have been identified within the tumor suppressor gene BRCA1. Most of them, called "variants of unknown significance" (VUS), cannot be classified as pathogenic or neutral by genetic methods, which complicates their cancer risk assessment. Functional assays have been developed to circumvent this uncertainty. They aim to determine how VUS impact the BRCA1 protein structure or function, thereby giving an indication of their potential to cause cancer. So far, three relevant assays have been designed in yeast and used on large sets of variants. However, they are limited to variants mapped in restricted domains of BRCA1. One of them, the small colony phenotype (SCP) assay, monitors the BRCA1-dependent growth of yeast colonies that increases with pathogenic but not neutral mutations positioned in the Cter region. Here, we extend this assay to the Nter part of BRCA1. We also designed a new assay, called the "yeast localization phenotype (YLP) assay," based on the accumulation of BRCA1 in a single inclusion body in the yeast nucleus. This phenotype is altered by variants positioned both in the Nter and Cter regions. Together, these assays provide new perspectives for the functional assessment of BRCA1 mutations in yeast.

Surface colonization underpins microbial ecology on terrestrial environments. Although factors that mediate bacteria–substrate adhesion have been extensively studied, their spatiotemporal dynamics during the establishment of microcolonies remains largely unexplored. Here, we use laser ablation and force microscopy to monitor single-cell adhesion during the course of microcolony formation. We find that adhesion forces of the rod-shaped bacteria Escherichia coli and Pseudomonas aeruginosa are polar. This asymmetry induces mechanical tension, and drives daughter cell rearrangements, which eventually determine the shape of the microcolonies. Informed by experimental data, we develop a quantitative model of microcolony morphogenesis that enables the prediction of bacterial adhesion strength from simple time-lapse measurements. Our results demonstrate how patterns of surface colonization derive from the spatial distribution of adhesive factors on the cell envelope., It is unclear how cell adhesion and elongation coordinate during formation of bacterial microcolonies. Here, Duvernoy et al. monitor microcolony formation in rod-shaped bacteria, and show that patterns of surface colonization derive from the spatial distribution of adhesive factors on the cell envelope.

Nucleosome positioning along eukaryotic genomes is crucial as it influences DNA accessibility for DNA binding proteins involved in DNA replication, transcription and repair. It is now accepted that both nucleosome preferences for some DNA sequences and remodeling factors play an important role in nucleosome positioning in vivo. However their relative importance remains a matter of debate. To investigate the role played by DNA sequence in nucleosome positioning in a cellular context we developped a strategy to assemble tandem DNA repeats of a nucleosome positioning sequence directly into Saccharomyces cerevisiae’s genome. This method is assisted by CRISPR/Cas9 and overlapping oligonucleotides and it turned out to be very efficient as it allowed to synthetize about 15 kilobases of tandem DNA repeats inside a yeast chromosome. Using this apporoach we obtained three yeast strains differing by the DNA linker length separating two consecutive monomeres of the 601 nucleosome positioning sequence. We chose three lengths of linker (20, 50 and 90 pb) for two reasons. First, they are compatible with the formation of a 30 nm chromatin fiber in vitro, and second, nucleosome repeat length of 167, 197 and 237 pb are found in eukaryotic genomes. We then verified if nucleosomes are effectively positioned according to the theoretic DNA linker lengths we designed in the “601” repeated region. To that goal we performed MNase-seq analysis to deduce nucleosomes dyads positions in the repeats. Interestingly our results show that the 601 sequence is not able to dictate strong nucleosomes positioning differing from the natural nucleosome repeat length of about 165 pb along the repeats in an in vivo context. We further investigated positions of dyads in the same three strains after inactivating the gene coding for the chromatin remodeler Chd1, which could potentially be responsible of the nucleosomes organization in the repeated area. Our results show no effect of Chd1, indicating that the “601” sequence has no positionning effect in vivo or that other trans-acting factors are implicated in nucleosome positioning in the engineered repeats. Finally, this work provides a new fast and simple approach for synthetic DNA repeats construction inside the yeast genome and could easily be applied for other synthetic chromatin engineering approaches., Le positionnement des nucléosomes le long des génomes eucaryotes est crucial étant donné qu’il affecte l’accessibilité de l’ADN à des protéines impliquées dans la transcription, la réplication, ou encore la réparation de l’ADN. Si il est aujourd’hui admis que les remodeleurs de chromatine ainsi que les préférences des nucléosomes pour certains motifs d’ADN constituent les deux principaux déterminants du positionnement des nucléosomes in vivo, leur importance relative fait encore l’objet de controverses. Dans le cadre de cette problématique nous avons développé une stratégie d’assemblage de répétitions de la séquence 601 positionnante de nucléosome directement dans le génome de Saccharomyces cerevisiae. Cette technique assistée par la technologie CRISPR/Cas9 et des oligonucléotides chevauchants s’est révélée très efficace et a permis d’assembler des répétitions sur une étendue d’environ 15 kilobases. Nous avons ainsi pu isoler trois souches se caractérisant par trois longueurs d’ADN de liaison de respectivement 20, 50 et 90 paires de bases séparant deux 601 consécutifs tout le long des répétitions. Ces longueurs d’ADN de liaison ont été choisies du fait de leur compatibilité avec les modèles de la fibre de 30 nm étudiés in vitro et parce qu’elles sont fréquemment observées chez les eucaryotes. Nous avons ensuite regardé si ces répétitions de la séquence 601 suffisent à dicter la succession des nucléosomes de S. cerevisiae selon le pas de chromatine attendu. Pour cela, nous avons eu recours à une approche de MNase-seq afin d’analyser les positions des dyades des nucléosomes dans les répétitions. Les résultats de ces analyses révèlent de façon intéressante l’incapacité de la séquence 601 à positionner le nucléosome dans ce contexte cellulaire et cela malgré l’étendue de la région de 601 répétés constituée. Nous avons également analysé le positionnement des nucléosomes chez ces trois mêmes souches suite à l’inactivation de Chd1, l’un des deux principaux architectes du paysage nucléosomal chez la levure, afin de s’affranchir de son potentiel effet sur le positionnement des nucléosomes dans la région 601. Nos résultats montrent que l’absence de Chd1 ne permet pas de rétablir un positionnement des nucléosomes sur les monomères de 601, suggérant que le 601 n’est pas positionnant in vivo ou que la région répétée est sous l’influence d’autres facteurs de remodelage. D’un point de vue méthodologique, notre technique de construction de répétitions in vivo permet d’envisager des approches simplifiées de biologie synthétique pour la construction de librairies de répétitions dans le génome de S. cerevisiae.