Quantitative electron microscopic analysis of DNA-protein interactions

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New possibilities in the observation of nucleic acids by electron spectroscopic imaging

The conditions for the use of Electron Spectroscopic Imaging (ESI) in the observation of DNA molecules adsorbed on carbon film are described. The Zeiss 902 electron microscope with its built-in spectrometer allows original modes of imaging, such as energy-filtered annular dark-field. The six different modes of imaging are illustrated with either positively stained or metallized DNA preparations to show the resulting important differences in contrast. The very strong contrasts obtained in annular or spectroscopic dark-field modes on specimens presenting a very low contrast in traditional bright-field are very useful for the observation of biological macromolecules. The advantages of some observation modes for imaging different types of preparations are discussed.Les conditions d'utilisation de l'imagerie par spectroscopie de pertes d'énergies des électrons pour l'observation de molécules d'ADN adsorbées sur film de carbone sont décrites. Le microscope électronique Zeiss 902 avec spectromètre incorporé à la colonne permet de former des images selon des modes originaux, tel que le fond noir annulaire filtré en énergie. Les six différents modes de formation des images sont illustrés sur des préparations d'ADN coloré positivement ou métallisé, pour montrer les importantes différences de contrastes. Les très forts contrastes obtenus en fond noir annulaire ou spectroscopique à partir de préparations ne présentant que très peu de contraste en fond clair traditionnel sont très utiles pour l'observation de macromolécules biologiques. Les avantages de certains modes d'observation pour l'étude de différents types de préparations sont discutés

Self-control in DNA site-specific recombination mediated by the tyrosine recombinase TnpI.

Tn4430 is a distinctive transposon of the Tn3 family that encodes a tyrosine recombinase (TnpI) to resolve replicative transposition intermediates. The internal resolution site of Tn4430 (IRS, 116 bp) contains two inverted repeats (IR1 and IR2) at the crossover core site, and two additional TnpI binding motifs (DR1 and DR2) adjacent to the core. Deletion analysis demonstrated that DR1 and DR2 are not required for recombination in vivo and in vitro. Their function is to provide resolution selectivity to the reaction by stimulating recombination between directly oriented sites on a same DNA molecule. In the absence of DR1 and/or DR2, TnpI-mediated recombination of supercoiled DNA substrates gives a mixture of topologically variable products, while deletion between two wild-type IRSs exclusively produces two-noded catenanes. This demonstrates that TnpI binding to the accessory motifs DR1 and DR2 contributes to the formation of a specific synaptic complex in which catalytically inert recombinase subunits act as architectural elements to control recombination sites pairing and strand exchange. A model for the organization of TnpI/IRS recombination complex is presented

Contribution of DNA Conformation and Topology in Right-handed DNA Wrapping by the Bacillus subtilis LrpC Protein

International audienceThe Bacillus subtilis LrpC protein belongs to the Lrp/AsnC family of transcriptional regulators. It binds the upstream region of the lrpC gene and autoregulates its expression. In this study, we have dissected the mechanisms that govern the interaction of LrpC with DNA by electrophoretic mobility shift assay, electron microscopy, and atomic force microscopy. LrpC is a structure-specific DNA binding protein that forms stable complexes with curved sequences containing phased A tracts and wraps DNA to form spherical, nucleosome-like structures. Formation of such wraps, initiated by cooperative binding of LrpC to DNA, results from optimal protein/protein interactions specified by the DNA conformation. In addition, we have demonstrated that LrpC constrains positive supercoils by wrapping the DNA in a right-handed superhelix, as visualized by electron microscopy