Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies

Abstract

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.

Beyond Sequence: Internucleosomal Interactions Dominate Array Assembly

“…Note that many structural studies of chromatin, including papers cited above, utilized repeats of the 601 motif . Furthermore, according to our recent paper, the 601 sequence has a considerably higher affinity for binding nucleosome cores. For a DNA substrate with 372 bp containing the 601 motif, the mononucleosomes are assembled at the position corresponding to the location of the 601 motif with a 98% preference.…”

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes

“…At the end of the DNA, opposite to the location of the 601 motif, we placed a three-way junction DNA segment forming a Y-shape, which served as the marker for mapping the nucleosomes on the DNA. 12 The nucleosomes were assembled as described in the methods section using a 2:1 molar ratio of the nucleosome core and DNA. The samples with CENP-A and canonical H3 nuc were assembled in parallel and prepared for AFM imaging as described earlier.…”

“…5 We used DNA templates with different sequences and AFM visualization to directly characterize the role of the DNA sequence on the positioning of nucleosomes and their interactions. 5,[9][10][11][12][13] In paper 13 , we used DNA templates with different sequences and found that nucleosomes are capable of close positioning with no discernible space between them, even in the case of assembled dinucleosomes. This array morphology contrasts with that observed for arrays assembled with repeats of the nucleosome positioning motifs separated by uniform spacers.…”