The EcoRI-DNA complex as a model for investigating protein-DNA interactions by atomic force microscopy.
Atomic force microscopy (AFM) is a technique widely used to image protein-DNA complexes, and its application has now been extended to the measurements of protein-DNA binding constants and specificities. However, the spreading of the protein-DNA complexes on a flat substrate, generally mica, is required prior to AFM imaging. The influence of the surface on protein-DNA interactions is therefore an issue which needs to be addressed. For that purpose, the extensively studied EcoRI-DNA complex was investigated with the aim of providing quantitative information about the surface influence. The equilibrium binding constant of the complex was determined by AFM at both low and high ionic strengths and compared to electrophoretic mobility shift assay measurements (EMSA). In addition, the effect of the DNA length on dissociation of the protein from its specific site was analyzed. It turned out that the AFM measurements are similar to those obtained by EMSA at high ionic strengths. We then advance the idea that this effect is due to the high counterion concentration near the highly negatively charged mica surface. In addition, a dissociation of the complexes once they are adsorbed onto the surface was observed, which is weakly dependent on the ionic strength contrary to what occurs in solution. Finally, a two-step mechanism, which describes the adsorption of the EcoRI-DNA complexes on the surface, is proposed. This model could also be extended to other protein-DNA complexes.
Sorel
Isabelle
I
Laboratoire Structures et Reconnaissance des Biomolécules, EA 3637, Université d'Evry, Rue du Père Jarlan, 91025 Evry Cedex, France.
Piétrement
Olivier
O
Hamon
Loïc
L
Baconnais
Sonia
S
Cam
Eric Le
EL
Pastré
David
D
D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D018625:Microscopy, Atomic Force; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D017931:DNA Primers; D004269:DNA, Bacterial; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015246:Deoxyribonuclease EcoRI