Skills (2)
Publications (38) View all
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Dataset: Supplementary to Conde
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Article: Kinetics of comet formation in single-cell gel electrophoresis: loops and fragments.
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ABSTRACT: We investigated the mechanisms of DNA exit during single-cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single-strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.Electrophoresis 01/2010; 31(3):512-9. · 3.30 Impact Factor -
Chapter: FLEXIBILITY OF NUCLEOSOMES ON TOPOLOGICALLY CONSTRAINED DNA
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ABSTRACT: The nucleosome plays an ever increasing role in our comprehension of the regulation of gene activity. Here we review our results on nucleosome conformational flexibility, its molecular mechanism and its functional relevance. Our initial approach combined both empirical measurement and theoretical simulation of the topological properties of single particles reconstituted on DNA minicircles. Two types of particles were studied in addition to the conventional nucleosome: a subnucleosome consisting of DNA wrapped around the (H3-H4)2 histone tetramer, now known as a tetrasome, and the linker histone H5/H1-bearing nucleosome, or chromatosome. All particles were found to thermally fluctuate between two to three conformational states, which differed by their topological and mechanical characteristics. These findings were confirmed for the nucleosome and the tetrasome by the use of magnetic tweezers to apply torsions to single arrays of these particles reconstituted on linear DNA. These latter experiments further revealed a new structural form of the nucleosome, the reversome, in which DNA is wrapped in a right-handed superhelical path around a distorted octamer. This work suggests that the single most important role of chromatin may be to considerably increase overall DNA flexibility, which might indeed be a requirement of genome function.01/2009: pages 251-291; -
Article: Nucleosome conformational flexibility and implications for chromatin dynamics: One contribution of 16 to a Theme 'The mechanics of DNA'
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ABSTRACT: The active role of chromatin in the regulation of gene activity seems to imply a conformational flexibility of the basic chromatin structural unit, the nucleosome. This review is devoted to our recent results pertaining to this subject, using an original approach based on the topology of single particles reconstituted on DNA minicircles, combined with their theoretical simulation. Three types of chromatin particles have been studied so far: a subnucleosome, that is, the (H3-H4)2 histone tetramer-containing particle, now known as the tetrasome; the nucleosome; and the linker histone H5/H1-bearing nucleosome (the chromatosome). All the particles were found to exist in two to three conformational states, which differ by their topological and mechanical properties. Our approach unveiled the molecular mechanisms of nucleosome conformational dynamics and will help to understand its functional relevance. A most surprising conclusion of the work was perhaps that DNA overall flexibility increases considerably upon particle formation, which might indeed be a requirement of genome function.Philosophical Transactions of The Royal Society B Biological Sciences 06/2004; 362:1519-1547. · 6.40 Impact Factor -
Article: Mechanisms of DNA exit during neutral and alkaline comet assay
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ABSTRACT: Results of the kinetics of DNA exit in neutral and alkaline variants of single cell gel electrophoresis are compared. It is shown that preincubation of samples in an alkaline buffer makes DNA exit in neutral electrophoresis impossible and, in contrast, effectively facilitates it within such buffers. We conclude that the alkali disrupts interactions between the loop domains of the DNA and the matrix proteins. We discuss the hypothetical nature of these interactions. The results obtained suggest that the mechanisms of DNA exit in the course of neutral and alkaline electrophoresis are substantially different. In the case of neutral electrophoresis, comet tails are formed by relaxed loop domains; during alkaline electrophoresis, they are formed by single-strand DNA fragments that are extracted, by the force of the electric field, from coils not yet bound to matrixes.Cytology and Genetics 04/2012; 43(6):367-370. · 0.25 Impact Factor
