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DNA-protein interactions

par Pierre-Louis Porté - 27 février 2007

Introduction

We study by fluorescence microscopy and at a single-molecule level the interactions between combed/stretched DNA and the type II restriction enzymes EcoRI and EcoRV. These enzymes play an important part in bacteria protection against viruses : they destroy viral DNA while cutting it in specific sites, preventing insertion and transcription in bacterial DNA. Biochemists have shown that restriction enzymes are able to locate their target with a remarkable effectiveness, which cannot be explained by a three-dimensional Brownian diffusion. A mechanism involving a linear diffusion of enzymes along DNA was proposed a few years ago (for a review, see N Shimamoto "One dimensional Diffusion of Proteins along DNA", J Biol. Chem 274, 1990). Direct observation of the dynamics of this process using fluorescence microscopy remains an experimental challenge. To achieve this goal, DNA molecules must be placed in a configuration which simultaneously allows observation and interaction with proteins. Proteins of interest must also be labeled with fluorophores while preserving activity.

Stretching DNA

In our experiments, the first step consists in unfolding the DNA molecules. For this purpose, we use two techniques :

Molecular combing : this method was developed at the ENS a few years ago (Bensimon A., Simon A, Chiffaudel A, Croquette V, Heslot F and Bensimon D., 1994. Science, 265). It consists in dipping a glass coverslip made hydrophobic in a solution at low pH containing DNA. DNA molecules bind by at least one of their ends to the surface. When the coverslip is withdrawn, molecules are aligned and uniformly overstretched by the receding meniscus. Molecules may be rehydrated but keep many points of contact with surface. DNA may also be combed by letting evaporate microdrops deposited on hydrophobic coverslips. During evaporation, DNA molecules are unfolded by the passage of the meniscus. Some enzymes can interact with combed DNA. Nevertheless, DNA-protein interactions are modified by the proximity of the surface and DNA overstretching. This is why we have developed a technique called DNA stretching :


Molecular combing	Combed DNA molecules

DNA stretching consists in stretching a DNA molecule by specifically attaching it by its ends to a surface. A solution containing DNA is injected into a flow cell. The binding of DNA ends is due to the low pH of the solution and stretching is obtained by application of an hydrodynamic flow in the cell. In this configuration, DNA is not overstretched and does not have other anchoring points on surface than its own ends. This configuration is more favorable than the one resulting from the combing in order to observe DNA-protein interactions. We have recently studied the transverse fluctuations of stretched DNA molecules.


DNA stretching obtained by the application of a hydrodynamic flow	Transverse fluctuations of stretched DNA molecules

These two techniques enable us to use Total Internal Reflection Fluorescence Microscopy (TIRFM) to observe interactions of enzymes with DNA. In contrast to epifluorescence microscopy, this technique limits the excitation by light to an area located in the vicinity of the surface ( 100 nm). Even if the enzymatic concentration is high, only the fluorescence of molecules located in the vicinity of the surface is collected. The signal-to-noise ratio is increased.

DNA-ends visualization

In order to visualize DNA using fluorescence microscopy, molecules are often tinted using an intercalating dye (YOYO) or a groove binding agent (SyBr). However, these dyes may interfere with enzymatic activity. Moreover, the photobleaching (destruction by light) damages the stretched/combed DNA, causing the break of the molecule. We have developed a technique which enables us to detect the ends of stretched/combed DNA molecules with quantum dots. These very brilliant fluorescent probes are not destroyed by light. The specificity and effectivness of this method offer a promising alternative to the usual DNA colouring in single-molecule experiments.

Combed DNA molecules on an hydrophobic surface. This ten kbp plasmid was tinted with intercalating dye YOYO1 (in green on the image) and revealed using quantum dots attached in a specific way to its ends (in red on the image).

Labeling enzymes with dyes

Another essential step consists in labeling enzymes with one or more fluorophore(s) to allow observation using fluorescence microscopy while preserving activity.

We work with biotinylated enzymes, kindly provided by Wolfgang Wende (team of A. Pingoud). These EcoRV enzymes are mutants for which a single cysteine (used for the grafting of the biotin), near to the active site in wild enzymes, has been moved in order to keep enzymatic activity. The biotinylated enzymes are then labeled with streptavidine-Cy3 or streptavidine-coated qdots (605nm).

Publications

"Transverse fluctuations of single DNA molecules attached at both extremities to a surface", A. Crut, D. Lasne, J.F. Allemand, M. Dahan, P. Desbiolles, Phys. Rev. E 67 051910 (2003)

"Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level", B. Géron-Landre, T. Roulon, P. Desbiolles and C. Escudé, Nucleic Acid Res. 31, e125 (2003).

"Detection of single DNA molecules by multicolor quantum-dot end-labeling", A. Crut, B. Géron-Landre, I. Bonnet, S. Bonneau, P. Desbiolles and C. Escudé, Nucl. Acid Res. 33 e98 (2005).