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Nucleosomes are stable complexes of DNA and histone proteins that are essential for the proper functioning of the genome. These structures must be unwrapped and disassembled for processes such as gene expression, replication, and repair. Histone post-translational modifications (PTMs) are known to play a significant role in regulating the structural changes of nucleosomes. However, the underlying mechanisms by which these modifications function remain unclear. In this study, we report the results of single molecule micromanipulation experiments on DNA–protein complexes composed of hyperacetylated histone proteins using transverse magnetic tweezers. The experiments were conducted by pre-extending λ-DNA with a force less than 4 $\mathrm{pN}$ before introducing hyperacetylated histones into the sample chamber. The DNA shortened as the histones formed complexes with it and the nucleosome arrays were then exposed to increasing tension, resulting in quantized changes in the DNA’s extension with step sizes of (integral multiples of) ~50 $\mathrm{nm}$. We also compared results of experiments using PTM histones and native histones with data collected for both types of histones for the same force ranges (2–80 $\mathrm{pN}$) and loading rates. Our data show that hyperacetylated nucleosomes require an unbinding force of around ~2.5 $\mathrm{pN}$, which is similar to that required for native histones. Moreover, we identified clear differences between the step-size distributions of native and hyperacetylated histones and found that in contrast to tethers reconstituted with native histones, the majority of nucleosomes in tethers compacted with hyperacetylated histones underwent disassembly at forces significantly lower than 6 $\mathrm{pN}$. Full article

We present a novel experimental setup in which magnetic and optical tweezers are combined for torque and force transduction onto single filamentous molecules in a transverse configuration to allow simultaneous mechanical measurement and manipulation. Previously we have developed a super-resolution imaging module which, in conjunction with advanced imaging techniques such as Blinking assisted Localisation Microscopy (BaLM), achieves localisation precision of single fluorescent dye molecules bound to DNA of ~30 nm along the contour of the molecule; our work here describes developments in producing a system which combines tweezing and super-resolution fluorescence imaging. The instrument also features an acousto-optic deflector that temporally divides the laser beam to form multiple traps for high throughput statistics collection. Our motivation for developing the new tool is to enable direct observation of detailed molecular topological transformation and protein binding event localisation in a stretching/twisting mechanical assay that previously could hitherto only be deduced indirectly from the end-to-end length variation of DNA. Our approach is simple and robust enough for reproduction in the lab without the requirement of precise hardware engineering, yet is capable of unveiling the elastic and dynamic properties of filamentous molecules that have been hidden using traditional tools. Full article