Rad51 Polymerization Reveals a New Chromatin Remodeling Mechanism

Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics

Binding of DNA to Natural Sepiolite: Applications in Biotechnology and Perspectives

International audienceDNA manipulation is crucial for many biotechnological prospects and for medical applications such as gene therapy. This requires the amplification and extraction of DNA from bacteria and the transfer of these DNA molecules into cells, including bacterial and mammalian cells. The capacity of the natural magnesium silicate clay mineral sepiolite to bind to DNA makes it a potential useful tool for biotechnological/medical strategies. In addition, sepiolite is inexpensive and classified as non-toxic and non-carcinogenic. This review will first describe the physico-chemical interactions between sepiolite and DNA. Then, the leverage of sepiolite/DNA interactions for DNA extraction from bacteria, to optimize DNA transfer into bacteria and DNA transfection into mammalian cells are presented. Finally, the putative toxicity of sepiolite and its advantages and perspectives for future prospects, such as the improvement of immunotherapy are also discussed

Microscopies moléculaires des complexes nucléoprotéiques

L’information génétique est portée dans chacune de nos cellules par environ deux mètres d’ADN enroulés au sein de larges assemblages macromoléculaires qui forment les chromosomes. Les microscopies moléculaires, électronique en transmission (TEM) et à force atomique (AFM), donnent accès à la résolution spatiale nécessaire pour imager et décrypter les mécanismes impliqués dans les grandes voies de régulation de la réparation, de la recombinaison, de la transcription et de la réplication du matériel génétique. Nous présentons ici quelques exemples de visualisation de machineries protéiques associées aux acides nucléiques

Study of the DNA/ethidium bromide interactions on mica surface by atomic force microscope: Influence of the surface friction

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Infrared nanospectroscopic imaging of DNA molecules on mica surface

International audienceAbstract Significant efforts have been done in last two decades to develop nanoscale spectroscopy techniques owning to their great potential for single-molecule structural detection and in addition, to resolve open questions in heterogeneous biological systems, such as protein–DNA complexes. Applying IR-AFM technique has become a powerful leverage for obtaining simultaneous absorption spectra with a nanoscale spatial resolution for studied proteins, however the AFM-IR investigation of DNA molecules on surface, as a benchmark for a nucleoprotein complexes nanocharacterization, has remained elusive. Herein, we demonstrate methodological approach for acquisition of AFM-IR mapping modalities with corresponding absorption spectra based on two different DNA deposition protocols on spermidine and Ni 2+ pretreated mica surface. The nanoscale IR absorbance of distinctly formed DNA morphologies on mica are demonstrated through series of AFM-IR absorption maps with corresponding IR spectrum. Our results thus demonstrate the sensitivity of AFM-IR nanospectroscopy for a nucleic acid research with an open potential to be employed in further investigation of nucleoprotein complexes

The EcoRI−DNA Complex as a Model for Investigating Protein−DNA Interactions by Atomic Force Microscopy

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Synthesis of a multi-branched porphyrin-oligonucleotide scaffold for the construction of DNA-Based nano-architecture

International audiencePorphytin-oligonudeotide hybrids containing one to four DNA strands (P1-P4) have been synthesised and fully characterised. The hybrid P4, which inserts one porphyrin and four DNA fragments, was combined with gold nanoparticles and images by electron microscopy

Core-shell polygalacturonate magnetic iron oxide nanoparticles: Synthesis, characterization, and functionalities

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Specific DNA−Protein Interactions on Mica Investigated by Atomic Force Microscopy

International audienceDNA processing by site-specific proteins on surface remains a challenging issue for nanobioscience applications and, in particular, for high-resolution imaging by atomic force microscopy (AFM). To obtain high-resolution conditions, mica, an atomically flat and negatively charged surface, is generally used. However, even though many specific DNA/protein interactions have already been observed by AFM, little is known about DNA accessibility to specific enzymes on mica. Here we measured the accessibility of adsorbed DNA to restriction endonucleases (EcoRI and EcoRV) using AFM. By increasing the concentration of divalent or multivalent salts, DNA adsorption on mica switches from weak to strong binding. Interestingly, while the accessibility of strongly bound DNA was inhibited, loosely adsorbed DNA was efficiently cleaved on mica. This result opens new perspective to study DNA/protein interaction by AFM or to modify specifically DNA on surface