January 1, 2016;
CENP-A and H3 Nucleosomes Display a Similar Stability to Force-Mediated Disassembly.
-specific nucleosomes are a central feature of the kinetochore
complex during mitosis, in which microtubules exert pulling and pushing forces upon the centromere
. CENP-A nucleosomes have been assumed to be structurally unique, thereby providing resilience under tension relative to their H3 canonical counterparts. Here, we directly test this hypothesis by subjecting CENP-A and H3 octameric nucleosomes, assembled on random or on centromeric DNA sequences, to varying amounts of applied force by using single-molecule magnetic tweezers. We monitor individual disassembly events of CENP-A and H3 nucleosomes. Regardless of the DNA sequence, the force-mediated disassembly experiments for CENP-A and H3 nucleosomes demonstrate similar rupture forces, life time residency and disassembly steps. From these experiments, we conclude that CENP-A does not, by itself, contribute unique structural features to the nucleosome that lead to a significant resistance against force-mediated disruption. The data present insights into the mechanistic basis for how CENP-A nucleosomes might contribute to the structural foundation of the centromere
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Fig 1. NAP1-assisted nucleosome assembly
(a) Schematic diagram of a tethered DNA molecule under tension in the magnetic tweezers assay. Torsionally unconstrained (nicked) DNA molecules were selected for nucleosome assembly. Assembly of a nucleosome results in shortening of the end-to-end length of the DNA. (b) Real-time observation of nucleosome assembly at 0.5 pN. Thin blue line shows raw data at 50 Hz bandwidth and thick blue line is the moving average with 1 s time window. During the flow of a buffer carrying histones and NAP1, the force was increased to 12 pN to suppress non-specific binding of the bead to the surface. After the flow, the force was lowered again to 0.5 pN and shortening of the DNA lengths was observed as nucleosomes assemble.
Fig 2. Real-time observation of nucleosome disassembly
(a) Model for the two-step process of the nucleosome disruption. Both outer- and inner-turn unwrapping processes release about 20–25 nm of the bound DNA. (b) A schematic diagram of nucleosome disassembly. Unwrapping of the DNA from a nucleosome results in a stepwise increase in the DNA length. (c) Representative force-extension curves measured with random DNA sequence before (black) and after (grey) the H3 nucleosome assembly. The force-extension curves were measured from 1 pN to 50 pN at a constant speed of 0.1 pN/s. After the assembly, multiple stepwise increases of the DNA extension were observed. Step sizes (in nm) determined from a step-finding algorithm indicated with a red vertical line next to each event highlighted. Inset: Magnification of a small region showing two discrete steps. (d) Representative force-extension curves measured with random DNA before (black) and after (grey) the CENP-A nucleosome assembly. (e-f) Step-size distributions of (e) H3 and (f) CENP-A nucleosome disassembly from random DNA (grey bars) and centromeric DNA (red bars). Blue lines indicate the 15–30 nm range expected for single-nucleosomal DNA-unwrapping events. Solid lines are Gaussian fits and the fit parameters are summarized in S1 Table. (g-h) Rupture-force distributions of the selected 15–30 nm steps from (g) H3 and (h) CENP-A nucleosome disassembly. Color scheme is the same as in (e). Solid lines are Gaussian fits to the data and the fit parameters are summarized in S2 Table.
Fig 3. Life time measurements of individual nucleosomes under a constant tension.
(a-b) Representative (a) H3 and (b) CENP-A nucleosome disassembly time trace from random DNA sequence under constant force. Red line is a most likely trace deduced by a step-finding algorithm. The determined step sizes (in nm) indicted next to each step. Top panel: the time trace of the force exerted on the DNA. At time 0, the force was increased abruptly from 1 pN to 30 pN. (c-d) Step-size distribution of (c) H3 and (d) CENP-A disassembly from random DNA (grey bars) and centromeric DNA (red bars). Blue lines indicates the 15–30 nm range expected from a single-nucleosome DNA-unwrapping event. Solid lines are multi-Gaussian fits and the fit parameters are summarized in S4 Table (e-f) Life time distributions of the selected 15–30 nm steps for (e) H3 and (f) CENP-A nucleosome disassembly. Color scheme is the same as (c). Solid lines are single-exponential fits to the data and the fit parameters are summarized in S5 Table. (g-h) Average life times of (g) H3 and (h) CENP-A nucleosomes obtained from single-exponential fits to the life-time distributions measured at different forces. Error bars are standard errors of the mean.
Andrews, The histone chaperone Nap1 promotes nucleosome assembly by eliminating nonnucleosomal histone DNA interactions. 2010, Pubmed