Chapter 7
Measurements of Protein–DNA Complexes Interactions by
Isothermal Titration Calorimetry (ITC) and Microscale
Thermophoresis (MST)
Amandine Gontier, Paloma F. Varela, Cle ´ ment Nemoz, Virginie Ropars,
Magali Aumont-Nicaise, Michel Desmadril, and
Jean-Baptiste Charbonnier
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 (K d ), the enthalpy (ΔH ),
and the stoichiometry N of an interaction. MST is used to measure the K d 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.
Key words Nucleo-protein complexes, Thermodynamic parameters, Microcalorimetry, Fluorescence,
Thermophoresis, Double-Strand Break repair, NHEJ
1 Introduction
The molecular mechanisms in the nucleus are regulated by intricate
networks of protein–protein and protein–DNA interactions. The
word “interaction” itself is used in the literature for many purposes.
It defines associations characterized by large-scale studies with
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Amandine Gontier and Paloma Ferna ´ndez Varela contributed equally with all other contributors.
125
Measurements of Protein–DNA Complexes Interactions by
Isothermal Titration Calorimetry (ITC) and Microscale
Thermophoresis (MST)
Amandine Gontier, Paloma F. Varela, Cle ´ ment Nemoz, Virginie Ropars,
Magali Aumont-Nicaise, Michel Desmadril, and
Jean-Baptiste Charbonnier
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 (K d ), the enthalpy (ΔH ),
and the stoichiometry N of an interaction. MST is used to measure the K d 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.
Key words Nucleo-protein complexes, Thermodynamic parameters, Microcalorimetry, Fluorescence,
Thermophoresis, Double-Strand Break repair, NHEJ
1 Introduction
The molecular mechanisms in the nucleus are regulated by intricate
networks of protein–protein and protein–DNA interactions. The
word “interaction” itself is used in the literature for many purposes.
It defines associations characterized by large-scale studies with
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Amandine Gontier and Paloma Ferna ´ndez Varela contributed equally with all other contributors.
125