During cell proliferation, newly replicated sister chromatids must be pulled apart and correctly positioned in opposite cell halves before division for faithful transmission of genetic information. In eukaryotes, segregation only occurs after replication has been fully achieved. The pairing of sister chromatids during S and G2 phase is termed Sister Chromatid Cohesion (SCC). SCC is mediated by the cohesins, a multi-subunit complex composed of Structural Maintenance of Chromosomes (SMC) proteins, which are deposited behind replication forks2. In contrast, replication and segregation are concomitant in bacteria. Bacterial chromosomes harbor a single origin of bidirectional replication, oriC, which defines two replication arms. As replication progresses along the two chromosomal arms, newly replicated loci migrate towards opposite cell halves. Pioneer microscopy observations showed that there is a lag of a few minutes between the time of replication of a locus and the segregation of the two resulting copies in Escherichia coli; the lag was subsequently associated with prolonged interactions between sister loci. Strictly speaking, bacteria lack an ortholog of cohesins. However, by analogy with eukaryotes, we will refer to this phenomenon as bacterial Sister Chromatid Cohesion (bSCC). Studies performed in different labs including the group of Partner 1 further suggested that segregation lags were in part due to the time Topo-IV took to remove catenation links (precatenanes) behind replication forks. We have recently demonstrated that bSCC becomes dependent on the induction of the recN gene product by the SOS response when DNA is damaged. Remarkably, RecN is a SMC protein, suggesting that its action might be similar to eukaryotic cohesins. The discovery of a first positive bSCC factor that extends cohesion in the presence of DNA damages suggests that, as SCC in eukaryotes, bSCC is an important aspect of the cell cycle that can be remodeled according to the growth conditions and influence the next events of this cycle. These observations led Partner 3 to develop a new tool, Hi-SC2, to survey bSCC at the genome level in different growth conditions. The BaCh project stems from our first Hi-SC2 results in V. cholera revealing that many cohesion factors are yet to be found, which might act at specific genomic positions, in specific environmental circumstances and by different mechanisms. The consortium pursues 3 objectives related to the characterization of basal post replicative and DNA damage induced induced cohesions: (1) characterise of bSSC factors in bacterial genomes, (2) characterise their mechanism of action at the molecular level and (3) determine the roles that cohesion might play in the bacterial cell cycle in normal or perturbed conditions. To reach these objectives BaCh will rely on state of the art genomics, genetics, live cell imaging and single molecule biophysics assays.Tools developped during BaCh should be highly valuable for the sisster chromatid cohesion field in various organisms. In addition, we believe that the BaCh project should help unmask primary overarching cohesion mechanisms, which are still at work in all cellular life forms. In this regard, comparison of E. coli and V. cholerae, two bacteria that share many potential bSCC factors but display very different DNA replication and segregation cycles, will be particularly informative.

The discovery of intrinsically disordered proteins and regions (IDPRs) challenges our understanding of the physical chemistry of biological mechanisms. IDPRs increase the reach of biomolecular systems to project far and engage in multiple interactions, moving efficiently over nanometer distances. Yet, we still miss methods to investigate the geometry and timescales of these nanometer motions with high resolution. We will develop an integrative experimental and computational framework to characterize nanometer motions in IDPRs at atomic resolution, exploiting synergies between paramagnetic NMR, electron paramagnetic resonance (EPR) and molecular dynamics (MD) simulations. A series of methodological innovations will be pursued in each of these fields: (1) we will tackle a key limitation that currently prohibits the quantitative interpretation of NMR relaxation effects due to the interaction with electrons by quantifying them over a broad range of the most relevant magnetic fields with a unique sample shuttle apparatus combined with high magnetic fields. (2) We will reduce the current flaws in molecular dynamics force fields for IDPs by direct improvement of the force fields and selection of MD trajectories based on experimental constraints with a new protocol. (3) The complementary information provided by paramagnetic NMR and EPR will allow us to carry an original quantitative analysis with MD simulations leading to an unprecedented description of the kinetics and conformational pathways of nanometer motions in IDPs with atomic resolution. This methodology will be developed on the IDPRs from key proteins in the non-homologous end joining (NHEJ) pathway, a process essential for the repair of DNA double-strand breaks and adaptive immunity: the long disordered region of the enzyme Artemis and the IDPRs of the scaffolding and regulation proteins XLF and XRCC4. The consortium brings together specialists in NMR methodology and instrumentation, IDP NMR, molecular dynamics simulations, protein EPR, and the biology of NHEJ. The NANO-DISPRO project will provide new tools to investigate the kinetic and thermodynamic principles that underlie the function of IDPRs.

Biological knowledge mostly comes from reference isolates from a few model organisms, which results in monomorphic descriptions of biological processes ignoring intra- and interspecific variations. Biodiversity within and among species has therefore been underappreciated while it harbors many resources of fundamental and biotechnological interests. Genomic exploration is notably essential to understand the genetic bases of phenotypic differences and therefore the patterns of molecular evolution, which is our purpose here. Different factors impact on genomic diversity among which meiotic recombination that affects the rate of introduction of new alleles combinations into populations and the genetic variability between populations. The long-term objective of RecombFun is to contribute to understand how genetic variation is created, maintained and generates phenotypic diversity, a main goal of evolutionary genetics. Our research proposal therefore consists of two ambitious and complementary axes. A population and functional genomics axis is dedicated to first, determine the evolution of the ancestral polymorphism and recombination patterns through the survey of the intraspecific genomic diversity across an entire subphylum, the Saccharomycotina yeasts. The second aspect of this population genomics axis is to dissect the variability of the genetic architecture of traits across species, a rather unexplored question so far. Here, we will bridge the gap between genetic variants and phenotypic diversity across different yeast species through QTL (Quantitative Trait Loci) and eQTL (expression Quantitative Trait Loci) mapping. In parallel, we will develop a molecular genomics axis to shed light on the control of meiotic recombination and its evolution. We will first determine experimentally the actual meiotic recombination landscapes within some of these non-conventional yeast species to determine the evolutionary fate of recombination hotspots and understand better their biology. Second, we will take advantage of the natural polymorphism in the genetic control of meiotic recombination to gain mechanistic insights. More specifically, we will exploit the absence of the interfering crossover pathway in the Lachancea yeast lineage to understand better the functional consequences of the presence and absence of this interfering pathway in closely related species. We will also try to decipher the mechanism that prevents recombination on an entire chromosome arm of the sex chromosome of Lachancea kluyveri, a phenomenon we recently discovered. Overall, RecomFun constitutes a remarkable example of the use of biodiversity to tackle questions dealing with mechanisms acting at both the molecular and cell population levels. There will be a high connectivity between the two axes since the same mapping populations will be used for both QTL mapping and determining the actual recombination maps. RecombFun is a multidisciplinary project involving two complementary teams who published last year a founding article (Brion et al. PLoS Genetics 2017). These teams combine a broad expertise ranging from the detailed mechanistic of recombination (Bertrand Llorente team) to the integration of the impact of recombination on the structure of natural populations (Joseph Schacherer team). They have strong expertise in bioinformatics and high-throughput approaches and strong publication records as underscored by two important articles published earlier this year (Peter et al. Nature 2018; Marsolier-Kergoat et al. Mol Cell 2018). This purely fundamental research project should bring significant breakthroughs in our understanding of the biology that lies between genotype and phenotype as well as of how genomes evolve, matters at the intersection of fundamental genetics, medicine and biotechnology, and therefore with strong societal impacts.