Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy

Fluorescence videomicroscopy and scanning force microscopy were used to fol low, in Peal 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 stro ng similarities to native chromatin fibers. In these extracts, the compacti on rate is more than 100 times faster than expected from standard biochemic al 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. T hey imply a sequential process with at least three steps, involving DNA wra pping as the final event. An absolute and quantitative measure of the kinet ic parameters of the early steps in chromatin assembly under physiological conditions could thus be obtained.