High-resolution biophysical analysis of the dynamics of nucleosome formation

Abstract

We describe a biophysical approach that enables changes in the structure of DNA to be followed during nucleosome formation in in vitro reconstitution with either the canonical “Widom” sequence or a judiciously mutated sequence. The rapid non-perturbing photochemical analysis presented here provides ‘snapshots’ of the DNA configuration at any given moment in time during nucleosome formation under a very broad range of reaction conditions. Changes in DNA photochemical reactivity upon protein binding are interpreted as being mainly induced by alterations in individual base pair roll angles. The results strengthen the importance of the role of an initial (H3/H4)2 histone tetramer-DNA interaction and highlight the modulation of this early event by the DNA sequence. (H3/H4)2 binding precedes and dictates subsequent H2A/H2B-DNA interactions, which are less affected by the DNA sequence, leading to the final octameric nucleosome. Overall, our results provide a novel, exciting way to investigate those biophysical properties of DNA that constitute a crucial component in nucleosome formation and stabilization.

Yeast HMO1: Linker Histone Reinvented

“…In vitro, nucleosome formation at a specific sequence is directed by intrinsic properties of the DNA, and it is nucleated by the association of the (H3/H4) 2 tetramer, which marks the initial point of DNA bending and therefore defines the dyad axis (Fig. 1A); the binding of (H3/H4) 2 is followed by the deposition of two H2A/H2B dimers (5)(6)(7). In vivo, nucleosome assembly is catalyzed by chaperones (8)(9)(10).…”

Nucleosome assembly and disassembly pathways

“…PhAST proved to be remarkably efficient in following structural changes in DNA as 601-and derived 601-nucleosomes were formed under decreasing ionic strength conditions, i.e. during nucleosome assembly [28]. We demonstrated first, the intuitive idea that histone binding induces noticeable structural changes in the local parameters of roll and twist all along the 601 sequence.…”

“…In view of this, we embarked on studies to capture nucleosome intermediate states during NaCl-induced assembly and disassembly (Schema 1) of the 601-nucleosome and to compare the two pathways. We used the PhAST (Photochemical Analysis of Structural Transitions) technique developed in our group [28].…”

“…This non-invasive technique can thus be applied to freely diffusing macromolecules in solution, without introducing structural artefacts caused for instance by bulky hydrophobic fluorophores [12]. It is based on the measurement of the probability to form, on the same DNA strand, UV photo-induced cyclobutane dimers between adjacent pyrimidines (YpY dimer, linked by C5-C5 and C6-C6 bonds) [28][29][30]. In addition to the quantum yields specific to each type of step, the probability of forming YpY dimers depends on the local YpY structure; more precisely on the inter base-pair parameters of roll and twist that are coupled in B-DNA in solution [31].…”

“…In addition to the quantum yields specific to each type of step, the probability of forming YpY dimers depends on the local YpY structure; more precisely on the inter base-pair parameters of roll and twist that are coupled in B-DNA in solution [31]. Thus, low twist and positive roll shorten the YpY C5-C5 and C6-C6 distances and thus favour dimer formation whereas large twist and negative roll have the inverse effect [28]. The YpY dimer probabilities along a DNA sequence therefore reflect its local average structure.…”