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Biophys J November 1, 2005; 89 (5): 3647-59.

Compaction kinetics on single DNAs: purified nucleosome reconstitution systems versus crude extract.

Wagner G , Bancaud A , Quivy JP , Clapier C , Almouzni G , Viovy JL .

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.

PubMed ID: 16100259
PMC ID: PMC1366857
Article link: Biophys J

References [+] :
Almouzni, Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs. 1988, Pubmed, Xenbase

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