Biophysical studies on Tn3 resolvase

Site-specific recombination is a process employed by organisms in order to perform spatially and temporally defined rearrangement of DNA molecules, such as phage integration and excision, resolution of circular multimers, inversions for expression of alternate genes, and assembly of genes during development. Tn3R is the prototype of a family of closely related mobile genetic elements referred to as the class II or Tn3 family of transposons. Tn3 contains the genes encoding a transposase, β-lactamase, and a site-specific recombinase, Tn3 resolvase (Tn3R), that is responsible for the resolution of the cointe-grate, an intermediate in the transposition reaction. Tn3R is able to resolve in vitro supercoiled plasmids containing two 114 bp res sites in direct orientation into two smaller circular plasmids, each of them with a single res site. In this thesis, the solution properties of Tn3R were studied by sedimentation equilibrium (SE) and velocity (SV) analytical ultracentrifugation and small angle neutron scattering (SANS). Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of kD1-2= 50 μM. SV and SANS demonstrated that the low-resolution conformation of dimeric Tn3R in solution is similar to that of γδ resolvase in the co-crystal structure by Yang and Steitz (1995), but with the DNA-binding domains in a rather extended conformation. In addition, equilibrium binding of Tn3R to the individual binding sites in res (sites I, II and III) was investigated by employing fluorescence anisotropy (FA) measurements. This revealed that site IIL (site II left end) and site III have the highest affinity for Tn3R, followed by site I, and finally by site HR (site II right end). The specificity of binding of Tn3R for non-specific DNA was assayed by competition experiments, where it was shown that the affinity of binding of Tn3R to site I is 1000 times higher than to non-specific sites. A new approach, involving a combination of rigid-body and ab initio modelling was developed for the study of the solution structure of macromolecules. At first, this approach was tested by applying it to the reconstruction of the low-resolution solution conformation of a DNA Holiday junction, based on small angle x-ray scattering and sedimentation velocity data. The scattering data were analysed in two independent ways: firstly, by rigid body modelling using previously suggested models for the Holliday junction (HJ), and secondly, by ab initio reconstruction methods. Sedimentation coefficients calculated for the models generated by both methods agreed with those determined experimentally and were compatible with the results of previous studies using different techniques, but provided a more direct and accurate determination of the solution conformation of the HJ. These results confirmed that addition of Mg2+ alters the conformation of the HJ from an extended to a stacked arrangement. The solution conformation of a stable protein-DNA complex formed by a mutant of Tn3R and DNA was studied by a similar approach. Hyperactive mutants of resolvase form a complex (X-synapse) containing two site I DNA fragments and a resolvase tetramer. The low-resolution solution structure of the purified, catalytically competent X-synapse was solved from small angle neutron and x-ray scattering data, by fitting the models constructed by rigid-body transformations of a published crystallographic structure of a resolvase dimer bound to site I to the data. This analysis revealed that the two site I fragments are on the outside of a resolvase tetramer core, and provided some information on the quaternary structure of the tetramer. Finally, the rigid-body modelling method was redesigned into a general systematic approach to retrieve the conformation of a macro molecule that simultaneously agrees with a range of experimental solution properties. In this method, generalised rigid-body modelling was combined with a Monte Carlo/simulated annealing optimisation method to search over a large range of possible conformations for the structure that best fits solution experimental properties derived from small angle scattering, fluorescence resonance energy transfer, and analytical ultracentrifugation datasets. This improved methodology was evaluated by applying it to two bulged DNA fragments with very different solution conformations

Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development

Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), for example, enhancers and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. In the present study, we employ Hi-M, a single-cell spatial genomics approach, to detect CRM–promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal that multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, before the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM–promoter interactions in defining transcriptional states, as well as distinct cell types.Fil: Espínola, Sergio Martín. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Université de Montpellier. Centre de Biologie Structurale; FranciaFil: Götz, Markus. Université de Montpellier. Centre de Biologie Structurale; Francia. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Bellec, Maelle. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; Francia. Université de Montpellier. Centre de Biologie Structurale; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Messina, Olivier. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; Francia. Université de Montpellier. Centre de Biologie Structurale; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Fiche, Jean Bernard. Université de Montpellier. Centre de Biologie Structurale; Francia. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Houbron, Christophe. Université de Montpellier. Centre de Biologie Structurale; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Centre National de la Recherche Scientifique; FranciaFil: Dejean, Matthieu. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; FranciaFil: Reim, Ingolf. Universitat Erlangen Nuremberg; AlemaniaFil: Cardozo Gizzi, Andres Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Alberto C. Taquini de Investigaciones en Medicina Traslacional - Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas "Prof. Dr. Alberto C. Taquini". Instituto Alberto C. Taquini de Investigaciones en Medicina Traslacional; ArgentinaFil: Lagha, Mounia. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; FranciaFil: Nollmann, Marcelo. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Université de Montpellier. Centre de Biologie Structurale; Franci

Challenges and guidelines toward 4D nucleome data and model standards

Due to recent advances in experimental and theoretical approaches, the dynamic three-dimensional organization (3D) of the nucleus has become a very active area of research in life sciences. We now understand that the linear genome is folded in ways that may modulate how genes are expressed during the basic functioning of cells. Importantly, it is now possible to build 3D models of how the genome folds within the nucleus and changes over time (4D). Because genome folding influences its function, this opens exciting new possibilities to broaden our understanding of the mechanisms that determine cell fate. However, the rapid evolution of methods and the increasing complexity of data can result in ambiguity and reproducibility challenges, which may hamper the progress of this field. Here, we describe such challenges ahead and provide guidelines to think about strategies for shared standardized validation of experimental 4D nucleome data sets and models

Publisher Correction: LifeTime and improving European healthcare through cell-based interceptive medicine.

A Correction to this paper has been published: https://doi.org/10.1038/s41586-021-03287-8.</jats:p

Biology across scales: from atomic processes to bacterial communities through the lens of the microscope

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Perspectives on Chromosome Organization

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