DNA substrate preparation for atomic force microscopy studies of protein–DNA interactions

Abstract

Protein-DNA interactions provide fundamental control mechanisms over biologically essential processes such as DNA replication, transcription, and repair. However, many details of these mechanisms still remain unclear. Atomic force microscopy (AFM) analyses provide unique and important structural and functional information on such protein-DNA interactions at the level of the individual molecules. The high sensitivity of the method with topographical visualization of all sample components also demands for extremely clean and pure materials. Here, we provide an overview of molecular biology-based approaches to produce DNA substrates for AFM imaging as well as other types of experiments, such as optical or magnetic tweezers, that profit from controllable substrate properties in long DNA fragments. We present detailed strategies to produce different types of motifs in DNA that are frequently employed targets of protein interactions. Importantly, the presented preparation techniques imply exact knowledge of the location of the introduced specific target sites within the DNA fragments, allowing for a distinction between specific and non-specific protein-DNA interactions in the AFM images and for separate conformational analyses of the different types of protein-DNA complexes.

Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility

“…The plasmid pUC19N (2,729 bp), kindly provided by S. Wilson's laboratory (NIEHS, National Institutes of Health), served as circular DNA for the AFM experiments and as the basis for the 916-bp linear specific DNA substrates. The linear DNA substrates for AFM experiments were prepared as described previously (29). Details are given in the supplemental materials.…”

“…For sample deposition, the incubations were diluted 8-fold in AFM deposition buffer (25 mM HEPES, pH 7.5, 25 mM sodium acetate, 10 mM magnesium acetate) to a final volume of 20 l and immediately pipetted onto freshly cleaved mica (Grade V; SPI Supplies), rinsed with ultra-pure deionized water and dried in a gentle stream of nitrogen. In the rinsing step, the negatively charged DNA polymers (Ϯ bound proteins) are stably chelated to the negative charges on the mica surface (at pH 7.5) by Mg 2ϩ ions in the applied buffer (29). Free protein molecules in the incubation also deposit and are fixed by the drying procedure on the substrate surface.…”

“…Free protein molecules in the incubation also deposit and are fixed by the drying procedure on the substrate surface. For the visualization of minute protein-DNA complexes, mica is superior as an AFM substrate to all other currently known materials (29). This layered silicate provides extremely clean, flat, and smooth surface properties with typical surface roughness of 0.05 nm (root mean square) and the further advantage of rapid and easy experimental sample preparation (29).…”

“…For the visualization of minute protein-DNA complexes, mica is superior as an AFM substrate to all other currently known materials (29). This layered silicate provides extremely clean, flat, and smooth surface properties with typical surface roughness of 0.05 nm (root mean square) and the further advantage of rapid and easy experimental sample preparation (29). AFM images were captured with a molecular force probe 3D-Bio AFM (Asylum Research, Santa Barbara, CA) in tapping mode using OMCL-AC240TS (Olympus) noncontact/tapping mode silicon probes with spring constants of ϳ2 N/m and resonance frequencies of ϳ75 kHz.…”

“…Enhanced binding of a protein to a specific target site within a DNA fragment results in a peak in the distribution at the corresponding position: 0% for complexes bound at DNA fragment ends, 50% for complexes bound at a DNA fragment center, ϳ31% for complexes bound at the lesion, and/or DNA bubble for specific DNA substrates in these experiments. Many proteins involved in DNA repair show a propensity to also bind to DNA fragment ends, likely as a result of local helix destabilization (29,32). XPD also shows a slight preference for DNA ends (bars at 0% DNA length in position distributions of Fig.…”