Abstract
Interactions between protein complexes and DNA are central regulators of the cell life. They control the activation and inactivation of a large set of nuclear processes including transcription, replication, recombination, repair, and chromosome structures. In the literature, protein–DNA interactions are characterized by highly complementary approaches including large-scale studies and analyses in cells. Biophysical approaches with purified materials help to evaluate if these interactions are direct or not. They provide quantitative information on the strength and specificity of the interactions between proteins or protein complexes and their DNA substrates. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) are widely used and are complementary methods to characterize nucleo-protein complexes and quantitatively measure protein–DNA interactions. We present here protocols to analyze the interactions between a DNA repair complex, Ku70–Ku80 (Ku) (154 kDa), and DNA substrates. ITC is a label-free method performed with both partners in solution. It serves to determine the dissociation constant (Kd), the enthalpy (ΔH), and the stoichiometry N of an interaction. MST is used to measure the Kd between the protein or the DNA labeled with a fluorescent probe. We report the data obtained on Ku–DNA interactions with ITC and MST and discuss advantages and drawbacks of both the methods.
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Acknowledgments
J.B.C. is supported by ARC program (SLS220120605310), ANR (ANR-12-SVSE8-012), INCA DomRep (PLBIO 2012-280), and CEFIPRA grant 5203C and by the French Infrastructure for Integrated Structural Biology (FRISBI) (ANR-10-INBS-05). A.G. is supported by a CIFRE PhD fellowship with Sanofi. We thank Pierre Soule from Nanotemper Technologies for his availability and all the fruitful discussion. The experiments were performed on the Platform PIM (Platform for measurements of Interactions of Macromolecules) (https://www.i2bc.paris-saclay.fr/spip.php?article280).
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Gontier, A. et al. (2021). Measurements of Protein–DNA Complexes Interactions by Isothermal Titration Calorimetry (ITC) and Microscale Thermophoresis (MST). In: Poterszman, A. (eds) Multiprotein Complexes. Methods in Molecular Biology, vol 2247. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1126-5_7
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DOI: https://doi.org/10.1007/978-1-0716-1126-5_7
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