Conclusive Evidence of the Reconstituted Hexasome Proven by Native Mass Spectrometry

Abstract

It has been suggested that the hexasome, in which one of the H2A/H2B dimers is depleted from the canonical nucleosome core particle (NCP), is an essential intermediate during NCP assembly and disassembly, but little structural evidence of this exists. In this study, reconstituted products in a conventional NCP preparation were analyzed by native electrospray ionization mass spectrometry, and it was found that the hexasome, which migrated in a manner almost identical to that of the octasome NCP in native polyacrylamide gel electrophoresis, was produced simultaneously with the octasome NCP. This result might contribute to understanding the assembly and disassembly mechanism of NCPs.

Dynamics of Nucleosome Assembly and Effects of DNA Methylation

“…A recent study confirmed that Nap1 dimer is a physiological conformation (54). On the contrary, hexasome existence has been confirmed based on a mass spectrometric analysis (55). The hexasome has been suggested as a physiological intermediate during nucleosome disassembly.…”

C‐terminal acidic domain of histone chaperone human NAP1 is an efficient binding assistant for histone H2A‐H2B, but not H3‐H4

“…; Heck ; Azegami et al . ) and compared them with the roles of yNAP1. We show that hNAP1 and yNAP1 dimers strongly and sequentially bind two H2A‐H2B heterodimers as first and second H2A‐H2B heterodimers by two different modes: exothermically and endothermically.…”

“…Here, we have investigated the roles of the core domain and CTAD of hNAP1 in binding to H2A-H2B and H3-H4 by isothermal calorimetry (ITC) and native mass spectrometry (MS) (Benesch et al 2007;Heck 2008;Azegami et al 2013) and compared them with the roles of yNAP1. We show that hNAP1 and yNAP1 dimers strongly and sequentially bind two H2A-H2B heterodimers as first and second H2A-H2B heterodimers by two different modes: exothermically and endothermically.…”

Direct Observation of H3–H4 Octasome by High‐Speed AFM

“…Here, it is demonstrated that ah istone H3-H4 octasome could be constructedw ithoutt he involvement of histoneH 2A-H2B under in vitro reconstitution conditions. [7] However,t he occurrenceo fs ub-nucleosomal and non-nucleosomals tructures like the hexasome, [8] and overlapping dinucleosome [9] formed by the dynamic environmenta re also known.Frequently occurring post-translational modification in H3 and H4 can affect histone-DNA interactions, which in turn destabilizet he nucleosome and facilitate histonee xchange. The octasome also displayed ao ne-step histone-dissociation pattern under heat treatment, distinct micrococcal nuclease and peplomycin accessibility,w hich suggests ad ifferent wrapping pattern to that in nucleosomes.…”

“…[1] The nucleosome comprises an octamero ff our core histone proteins (H3, H4, H2A, and H2B) and a1 45 to 147-base pair (bp) segment of DNA, [2][3][4] Histone H3 and H4 proteins act as the precursor in nucleosome reconstitution and orientatet he DNA sequencet of orm at etrasome, the intermediate stage in the nucleosome formation. [7] However,t he occurrenceo fs ub-nucleosomal and non-nucleosomals tructures like the hexasome, [8] and overlapping dinucleosome [9] formed by the dynamic environmenta re also known. [7] However,t he occurrenceo fs ub-nucleosomal and non-nucleosomals tructures like the hexasome, [8] and overlapping dinucleosome [9] formed by the dynamic environmenta re also known.…”