For over 25 years, imaging of DNA by atomic force microscopy has been intensely pursued. Ideally, such images are then used to probe the physical properties of DNA and characterize protein–DNA interactions. The atomic flatness of mica makes it the preferred substrate for high signal-to-noise ratio (SNR) imaging, but the negative charge of mica and DNA hinders deposition. Traditional methods for imaging DNA and protein–DNA complexes in liquid have drawbacks: DNA conformations with an anomalous persistence length (p), low SNR, and/or ionic deposition conditions detrimental to preserving protein–DNA interactions. Here, we developed a process to bind DNA to mica in a buffer containing both MgCl2 and KCl that resulted in high SNR images of equilibrated DNA in liquid. Achieving an equilibrated 2D configuration (i.e., p = 50 nm) not only implied a minimally perturbative binding process but also improved data quality and quantity because the DNA’s configuration was more extended. In comparison to a purely NiCl2-based protocol, we showed that an 8-fold larger fraction (90%) of 680-nm-long DNA molecules could be quantified. High-resolution images of select equilibrated molecules revealed the right-handed structure of DNA with a helical pitch of 3.5 nm. Deposition and imaging of DNA was achieved over a wide range of monovalent and divalent ionic conditions, including a buffer containing 50 mM KCl and 3 mM MgCl2. Finally, we imaged two protein–DNA complexes using this protocol: a restriction enzyme bound to DNA and a small three-nucleosome array. We expect such deposition of protein–DNA complexes at biochemically relevant ionic conditions will facilitate biophysical insights derived from imaging diverse protein–DNA complexes.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that methylates histone H3 at Lysine 27. PRC2 is critical for epigenetic gene silencing, cellular differentiation and the formation of facultative heterochromatin. It can also promote or inhibit oncogenesis. Despite this importance, the molecular mechanisms by which PRC2 compacts chromatin are relatively understudied. Here, we visualized the binding of PRC2 to naked DNA in liquid at the single-molecule level using atomic force microscopy. Analysis of the resulting images showed PRC2, consisting of five subunits (EZH2, EED, SUZ12, AEBP2 and RBBP4), bound to a 2.5-kb DNA with an apparent dissociation constant ($K_{\rm{D}}^{{\rm{app}}}$) of 150 ± 12 nM. PRC2 did not show sequence-specific binding to a region of high GC content (76%) derived from a CpG island embedded in such a long DNA substrate. At higher concentrations, PRC2 compacted DNA by forming DNA loops typically anchored by two or more PRC2 molecules. Additionally, PRC2 binding led to a 3-fold increase in the local bending of DNA’s helical backbone without evidence of DNA wrapping around the protein. We suggest that the bending and looping of DNA by PRC2, independent of PRC2’s methylation activity, may contribute to heterochromatin formation and therefore epigenetic gene silencing.

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