Abstract
Nucleosome assembly is a vital part of chromatin maintenance for all eukaryotic cells. The histone chaperone NAP1 is one of the proteins involved in this process, guiding histones into the nucleus and assembling them into nucleosomes without use of ATP or other energy sources. It is known that NAP1 first brings H3H4 histone tetramers to the DNA to form a tetrasome, and in a second step H2a and H2B histones are added, but mechanistic insight into this process is still lacking. We use high-speed AFM to image NAP-1 assembled tetrasomes in vitro with spatial and temporal resolutions of the order of 1 nm and 1 s. We observe a rich palette of dynamical processes, among which are spontaneous tetrasome dissociation, cluster formation and a transient association between bare DNA and NAP1. One intriguing observation is the hopping of a tetrasome between two stable positions spaced only a few nanometers apart. We believe this motion can be ascribed to a re-orientation of the DNA around the histones, a phenomenon that we also observed in magnetic tweezer single molecule assays.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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