Abstract
While many proteins are involved in the assembly and (re)positioning of nucleosomes, the dynamics of protein-assisted nucleosome formation are not well understood. We study NAP1 (nucleosome assembly protein 1) assisted nucleosome formation at the single-molecule level using magnetic tweezers. This method allows to apply a well-defined stretching force and supercoiling density to a single DNA molecule, and to study in real time the change in linking number, stiffness and length of the DNA during nucleosome formation. We observe a decrease in end-to-end length when NAP1 and core histones (CH) are added to the dsDNA. We characterize the formation of complete nucleosomes by measuring the change in linking number of DNA, which is induced by the NAP1-assisted nucleosome assembly, and which does not occur for non-nucleosomal bound histones H3 and H4. By rotating the magnets, the supercoils formed upon nucleosome assembly are removed and the number of assembled nucleosomes can be counted. We find that the compaction of DNA at low force is about 56 nm per assembled nucleosome. The number of compaction steps and associated change in linking number indicate that NAP1-assisted nucleosome assembly is a two-step process.
Highlights
IntroductionDNA is wrapped around histone octamers, forming nucleosomes [1]
In eukaryotic cells, DNA is wrapped around histone octamers, forming nucleosomes [1]
Protein-induced length decrease of DNA We examine the real time nucleosome assembly protein1 (NAP1)-assisted assembly of core histones onto DNA by continuously monitoring the end-to-end length of single DNA molecules upon addition of histones and NAP1 (Figure 2)
Summary
DNA is wrapped around histone octamers, forming nucleosomes [1]. DNA is wrapped 1.7 times around a disklike octamer which is built up from eight histones; two copies each of H2A, H2B, H3 and H4, all of which have a positive charge. Histones and the DNA do not spontaneously self-assemble into nucleosomes [4], but form large aggregates. Chaperones bring the histones to the DNA in the correct order. Several histone chaperones, such as chromatin assembly factor 1 (CAF1) [8], HIRA [9], and nucleosome assembly protein (NAP1) [10] have been characterized as chromatin assembly factors
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