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CENP-A is a histone variant found in high abundance at the centromere in humans. At the centromere, this histone variant replaces the histone H3 found throughout the bulk chromatin. Additionally, the centromere comprises tandem repeats of α-satellite DNA, which CENP-A nucleosomes assemble upon. However, the effect of the DNA sequence on the nucleosome assembly and centromere formation remains poorly understood. Here, we investigated the structure of nucleosomes assembled with the CENP-A variant using Atomic Force Microscopy. We assembled both CENP-A nucleosomes and H3 nucleosomes on a DNA substrate containing an α-satellite motif and characterized their positioning and wrapping efficiency. We also studied CENP-A nucleosomes on the 601-positioning motif and non-specific DNA to compare their relative positioning and stability. CENP-A nucleosomes assembled on α-satellite DNA did not show any positional preference along the substrate, which is similar to both H3 nucleosomes and CENP-A nucleosomes on non-specific DNA. The range of nucleosome wrapping efficiency was narrower on α-satellite DNA compared with non-specific DNA, suggesting a more stable complex. These findings indicate that DNA sequence and histone composition may be two of many factors required for accurate centromere assembly. 

Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes. 

The interplay between the mechanical properties of double-stranded and single-stranded DNA is a phenomenon that contributes to various genetic processes in which both types of DNA structures coexist. Highly stiff DNA duplexes can stretch single-stranded DNA (ssDNA) segments between the duplexes in a topologically constrained domain. To evaluate such an effect, we designed short DNA nanorings in which a DNA duplex with 160 bp is connected by a 30 nt single-stranded DNA segment. The stretching effect of the duplex in such a DNA construct can lead to the elongation of ssDNA, and this effect can be measured directly using atomic force microscopy (AFM) imaging. In AFM images of the nanorings, the ssDNA regions were identified, and the end-to-end distance of ssDNA was measured. The data revealed a stretching of the ssDNA segment with a median end-to-end distance which was 16% higher compared with the control. These data are in line with theoretical estimates of the stretching of ssDNA by the rigid DNA duplex holding the ssDNA segment within the nanoring construct. Time-lapse AFM data revealed substantial dynamics of the DNA rings, allowing for the formation of transient crossed nanoring formations with end-to-end distances as much as 30% larger than those of the longer-lived morphologies. The generated nanorings are an attractive model system for investigation of the effects of mechanical stretching of ssDNA on its biochemical properties, including interaction with proteins. 

Abstract Chromatin structure is dictated by nucleosome assembly and internucleosomal interactions. The tight wrapping of nucleosomes inhibits gene expression, but modifications to histone tails modulate chromatin structure, allowing for proper genetic function. The histone H4 tail is thought to play a large role in regulating chromatin structure. Here we investigated the structure of nucleosomes assembled with a tail-truncated H4 histone using Atomic Force Microscopy. We assembled tail-truncated H4 nucleosomes on DNA templates allowing for the assembly of mononucleosomes or dinucleosomes. Mononucleosomes assembled on nonspecific DNA led to decreased DNA wrapping efficiency. This effect is less pronounced for nucleosomes assembled on positioning motifs. Dinucleosome studies resulted in the discovery of two effects- truncation of the H4 tail does not diminish the preferential positioning observed in full-length nucleosomes, and internucleosomal interaction eliminates the DNA unwrapping effect. These findings provide insight on the role of histone H4 in chromatin structure and stability.