Abstract
The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates, explained in current models by a combination of 3D and 1D diffusion. Here, we present a review of the single-molecule approaches at the forefront of the study of protein-DNA interaction dynamics and target search in vitro and in vivo. Flow stretch, optical and magnetic manipulation, single fluorophore detection and localization as well as combinations of different methods are described and the results obtained with these techniques are discussed in the framework of the current facilitated diffusion model.
Highlights
At the most elemental level, all DNA biological functions are carried out by individual proteins that must interact with DNA to trigger molecular processes indispensable to the cell
DNA is confined in a cellular compartment, and the accessibility of DNA sequences to proteins is further restricted by the supercoiled structure of native DNA in eubacteria or by nucleosomes in eukaryotic chromatin
The disassembly process resulting from the interplay between tension-independent ATP hydrolysis and the release of the tension stored in the filament at a force-dependent rate could only be revealed thanks to the simultaneous monitoring of the fluorescence signal from the protein and the tension of the DNA [157]. Another recent study reports the combination of single-molecule fluorescence and 2OTs to directly visualize the sliding of restriction enzyme EcoRV labeled with a single Quantum dot (QD) along a DNA molecule held by 2OTs [158]
Summary
At the most elemental level, all DNA biological functions are carried out by individual proteins that must interact with DNA (usually with specific sequences) to trigger molecular processes indispensable to the cell. As a result of these studies, the facilitated diffusion model [3,16,17,18,19,20,21] is currently the most accepted theory of DNA target searching, and it has been recently demonstrated in vivo for LacI [22]. The complex interplay of all these processes determines the rate at which a protein can scan through the excess non-specific DNA and find the needle in the haystack, i.e., its target Some of these processes, for example intersegmental transfer, require specific structural features such as the ability of binding simultaneously to multiple sites, but all of the most basic properties of the search process can be described in terms of association and dissociation rate constants (as distinguished between those relative to specific and non-specific DNA) and diffusion constants (both 3D and 1D). In Vitro Monitoring of Facilitated Diffusion and Protein-DNA Interactions at the Single-Molecule Level
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