DNA bending induced by the archaebacterial histone-like protein MC1.
The conformational changes induced by the binding of the histone-like protein MC1 to DNA duplexes have been analyzed by dark-field electron microscopy and polyacrylamide gel electrophoresis. Visualisation of the DNA molecules by electron microscopy reveals that the binding of MC1 induces sharp kinks. Linear DNA duplexes (176 bp) which contained a preferential site located at the center were used for quantitative analysis. Measurements of the angle at the center of all duplexes, at a fixed DNA concentration, as a function of the MC1 concentration, were very well fitted by a simple model of an isotropic flexible junction and an equilibrium between the two conformations of DNA with bound or unbound MC1. This model amounts to double-folded Gaussian distributions and yields an equilibrium deflection angle of theta0=116 degrees for the DNA with bound MC1. It allowed measurements of the fraction of DNA with bound MC1 to be taken as a function of MC1 concentrations and yields an equilibrium dissociation constant of Kd=100 nM. It shows that the flexibility of DNA is reduced by the binding of MC1 and the formation of a kink. The equilibrium dissociation constant value was corroborated by gel electrophoresis. Control of the model by the computation of the reduced chi2 shows that the measurements are consistent and that electron microscopy can be used to quantify precisely the DNA deformations induced by the binding of a protein to a preferential site.
Cam
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EL
Laboratoire de Microscopie Moléculaire et Cellulaire, LM2C, UMR 1772 CNRS, Institut Gustave-Roussy, 39 rue Camille Desmoulins, Villejuif Cedex, 94805, France. elecam@igr.fr
Culard
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F
Larquet
E
E
Delain
E
E
Cognet
J A
JA
D019843:Archaeal Proteins / Q000648:ultrastructure*; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D007091:Image Processing, Computer-Assisted; D017020:Methanosarcina / Q000235:genetics*; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012261:Ribonucleoproteins / Q000648:ultrastructure*
D019843:Archaeal Proteins; D004269:DNA, Bacterial; C051211:HMb protein, Methanosarcina; D012261:Ribonucleoproteins