Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy.
Fluorescence videomicroscopy and scanning force microscopy were used to follow, in real time, chromatin assembly on individual DNA molecules immersed in cell-free systems competent for physiological chromatin assembly. Within a few seconds, molecules are already compacted into a form exhibiting strong similarities to native chromatin fibers. In these extracts, the compaction rate is more than 100 times faster than expected from standard biochemical assays. Our data provide definite information on the forces involved (a few piconewtons) and on the reaction path. DNA compaction as a function of time revealed unique features of the assembly reaction in these extracts. They imply a sequential process with at least three steps, involving DNA wrapping as the final event. An absolute and quantitative measure of the kinetic parameters of the early steps in chromatin assembly under physiological conditions could thus be obtained.
Ladoux
B
B
Laboratoire de Physico-Chimie Curie (Unité Mixte de Recherche Centre National de la Recherche Scientifique/Institut Curie 168), Paris Cedex, France.
Quivy
J P
JP
Doyle
P
P
du Roure
O
O
Almouzni
G
G
Viovy
J L
JL
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D002457:Cell Extracts; D002843:Chromatin / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004330:Drosophila; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D008958:Models, Molecular; D009707:Nucleosomes / Q000378:metabolism*; D013997:Time Factors; D014981:Xenopus
D002457:Cell Extracts; D002843:Chromatin; D004279:DNA, Viral; D009707:Nucleosomes