Compaction kinetics on single DNAs: purified nucleosome reconstitution systems versus crude extract
Gaudeline Wagner 1, Aurélien Bancaud 1, Jean-Pierre Quivy 2, Cédric Clapier 3, Geneviève Almouzni 2 and Jean-Louis Viovy 1*
1 Institut Curie UMR 168
2 Institut Curie UMR 218
3 EMBL Grenoble
* To whom correspondence should be addressed. E-mail: jean-louis.viovy{at}curie.fr.
Submitted on March 14, 2005
Revised on April 20, 2005
Accepted on 5 July 2005
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Abstract |
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Kinetics of compaction on single DNA molecules are studied by fluorescence videomicroscopy in the presence of (i) Xenopus egg extracts and (ii) purified nucleosome reconstitution systems using a combination of histones with either the histone chaperone yNAP-1, or negatively charged macromolecules, such as polyglutamic acid (PGA) and RNA. The comparison shows that the compaction rates can differ by up to a factor 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 yNAP-1 increases both the rate of the reaction and the packing ratio of the final product. These stimulatory effects could not be obtained with PGA 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.
Key Words:
DNA-protein interaction, fluorescence videomicroscopy, histone-chaperone interaction, real-time kinetics analysis, single molecule biophysics
