title: Compaction kinetics on single DNAs: purified nucleosome reconstitution systems versus crude extract.
abstract: 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.
authors.affiliation: Laboratoire PhysicoChimie Curie, Institut Curie, CNRS UMR 168, 75248 Paris, France.
authors.forename: Gaudeline
authors.initials: G
authors.lastname: Wagner
authors.forename: Aurélien
authors.initials: A
authors.lastname: Bancaud
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Quivy
authors.forename: Cédric
authors.initials: C
authors.lastname: Clapier
authors.forename: Geneviève
authors.initials: G
authors.lastname: Almouzni
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Viovy
mesh_terms: D000818:Animals; D000838:Anions; D001710:Biotin / Q000737:chemistry; D018797:Cell Cycle Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism*; D002843:Chromatin / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004330:Drosophila; D029721:Drosophila Proteins; D006657:Histones / Q000737:chemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D015233:Models, Statistical; D009687:Nuclear Proteins / Q000737:chemistry; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000737:chemistry; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D013997:Time Factors; D014981:Xenopus; D016875:tau Proteins / Q000737:chemistry
chemical_list: D000838:Anions; D018797:Cell Cycle Proteins; D002843:Chromatin; D018076:DNA, Complementary; D029721:Drosophila Proteins; D006657:Histones; C533554:Nap1 protein, Drosophila; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes; D016875:tau Proteins; D011099:Polyglutamic Acid; D012313:RNA; D001710:Biotin; D004247:DNA; D019809:Streptavidin