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Compaction Kinetics on Single DNAs: Purified Nucleosome Reconstitution Systems versus Crude Extract

Gaudeline Wagner ^*, Aurélien Bancaud ^*, Jean-Pierre Quivy , Cédric Clapier , Geneviève Almouzni  and Jean-Louis Viovy ^*

^* Laboratoire PhysicoChimie Curie, Institut Curie, Paris, France; Laboratoire Dynamique et Plasticité du Génome, Institut Curie, Paris, France; and European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France

Correspondence: Address reprint requests to Jean-Louis Viovy, Laboratoire PhysicoChimie Curie, Institut Curie, CNRS UMR 168, Paris, 75248 France. Tel.: 033-1-42-34-67-52; E-mail: Jean-Louis.Viovy{at}curie.fr.

Kinetics of compaction on single DNA molecules are studied by fluorescence videomicroscopy in the presence of 1), Xenopus egg extracts and 2), purified nucleosome reconstitution systems using a combination of histones with either the histone chaperone Nucleosome Assembly Protein (NAP-1) or negatively charged macromolecules such as polyglutamic acid and RNA. The comparison shows that the compaction rates can differ by a factor of up to 1000 for the same amount of histones, depending on the system used and on the presence of histone tails, which can be subjected to post-translational modifications. Reactions with purified reconstitution systems follow a slow and sequential mechanism, compatible with the deposition of one (H3-H4)₂ tetramer followed by two (H2A-H2B) dimers. Addition of the histone chaperone NAP-1 increases both the rate of the reaction and the packing ratio of the final product. These stimulatory effects cannot be obtained with polyglutamic acid or RNA, suggesting that yNAP-1 impact on the reaction cannot simply be explained in terms of charge screening. Faster compaction kinetics and higher packing ratios are reproducibly reached with extracts, indicating a role of additional components present in this system. Data are discussed and models proposed to account for the kinetics obtained in our single-molecule assay.