Quantitative electron microscopic analysis of DNA‐protein interactions

Abstract

Electron microscopy offers a unique potentiality to visualize individual molecules. For the last 30 years it has been used to study the structure and the interactions of various biological macromolecules. The contribution of electron microscopy is important because of its capacity to demonstrate the existence of conformational structures such as kinks, bents, loops, etc., either on naked DNA, or on DNA associated with various proteins or ligands. Increasing interest was given to such observations when it was found that they provide a direct visualization of interacting molecules involved in DNA metabolism and gene regulation. Technical advances in the preparation of the specimens, their observation in the electron microscope, and the image processing by computers have allowed the shifting from qualitative to quantitative analysis, as illustrated by a few examples from our laboratory.

Transmission Electron Microscopy Reveals an Optimal HIV-1 Nucleocapsid Aggregation with Single-stranded Nucleic Acids and the Mature HIV-1 Nucleocapsid Protein

“…[52][53][54][55] By using this technique, the data presented here clearly emphasize profound differences in the NCp7-directed aggregation of ssDNA (similar to RNA) versus dsDNA, especially in an RT-optimized medium. The results also highlight a critical difference in binding properties between NCp7 and its precursor NCp15, and correlates with recent results showing the failure of aggregating properties of NCp15 with DNA.…”

Human Immunodeficiency Virus Type 1 Central DNA Flap: Dynamic Terminal Product of Plus-Strand Displacement DNA Synthesis Catalyzed by Reverse Transcriptase Assisted by Nucleocapsid Protein

“…To obtain a good spreading of ssDNA, T4 SSB gp32 was added to the DNA solution at a ratio of one gp32 tetramer per 5 nt. Electron microscopy (EM) was performed as described previously (30,31). First, 5 l of a DNA solution, at a final concentration of 1g/ml, was spotted onto a 600-mesh copper grid coated with a very thin carbon film activated by a glow discharge in the presence of pentylamine according to the method of Dubochet et al (10).…”

Observation of binding and polymerization of Fur repressor onto operator-containing DNA with electron and atomic force microscopes.

“…Analysis of DNA-protein interactions by EM provides original information, complementary to that obtained with conventional biochemical methods (6)(7)(8). Mapping of DNA-protein interactions and of either intrinsic or induced local conformational variations of DNA is possible, as previously described (7)(8)(9). Furthermore, negative staining provides additional information about the structural organization of proteins bound to DNA.…”