Compaction kinetics on single DNAs: purified nucleosome reconstitution systems versus crude extract.
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.
Wagner
Gaudeline
G
Laboratoire PhysicoChimie Curie, Institut Curie, CNRS UMR 168, 75248 Paris, France.
Bancaud
Aurélien
A
Quivy
Jean-Pierre
JP
Clapier
Cédric
C
Almouzni
Geneviève
G
Viovy
Jean-Louis
JL
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
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