two-hybrid or tap-tag approaches and also in cell analyses with
proximity ligation assays (PLA) or co-immunoprecipitation [1, 2]. “Interactions” include also genetic analyses as well as
biophysical approaches and structural studies of complexes. This
large set of approaches describe the same event of “interaction”
and leads in some case to controversy. On the contrary, these
multiple analyses favor in many cases an integrate view of the
relevance of a given protein complex both in vitro and in the cell.
For example, protein interaction measurements in the cellular context provide a characterization of the interactions in the presence of
potential competitors or of post-translational modifications at different steps of the cell cycle. Biophysical approaches nicely complement these data by confirming or not the direct interaction with
purified proteins and ligands. Biophysical measurements are central
to characterize the strength, the specificity, and the stoichiometry
of the interactions in a quantitative manner. Interactions measured
in cells and not confirmed in vitro can also be informative of the
role of an additional partner or the need for post-translational
modifications eventually absent on purified proteins used for in vitro
studies.
A large panel of biophysical approaches are used nowadays to
perform quality controls and interaction analyses on macromolecular samples (some of these are presented in other chapters of this
issue). Each methodology offers complementary information and
comes with specific requirements on sample preparation. We present here protocols for Isothermal Titration Calorimetry (ITC) and
MicroScale Thermophoresis (MST) analyses of the interactions
between a protein complex and DNA substrates. The complex is
formed by the heterodimer Ku70–Ku80 (or Ku). Ku is a core factor
of the main DNA Double-Strand Break (DSB) repair pathway in
human, called Non-Homologous End-Joining (NHEJ). Ku recognizes the DSB ends through its ring-shaped structure [3]. The
heterodimer has a high affinity, in the nanomolar range, for
double-strand DNA (15 bp minimum). The K d of the Ku–DNA
interaction was initially characterized by gel shift assays (EMSA) [4]
and fluorescent anisotropy analyses [5]. Ku binds DNA in a
sequence-independent manner and in vitro one Ku molecule can
thread on DNA with one Ku bound every 15–20 bp of DNA
duplex [3, 6]. Interestingly, a super resolution microscopy study
showed that in cells only one or two Ku molecules bind to DSB
ends, suggesting a limited threading of Ku from the DNA DSB
ends in a cellular context [7]. Ku plays also a central role for the
recruitment of the enzymatic activities (nucleases, polymerases,
ligases) that process and ligate the DSB ends [8, 9]. Our laboratory
recently described at the molecular level the mechanism of recruitment by Ku of some of the downstream NHEJ factors [10]. Here,
we present the measurements by ITC of the thermodynamic parameters of the interaction between Ku and DNA substrates, and we
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Amandine Gontier et al.
proximity ligation assays (PLA) or co-immunoprecipitation [1, 2]. “Interactions” include also genetic analyses as well as
biophysical approaches and structural studies of complexes. This
large set of approaches describe the same event of “interaction”
and leads in some case to controversy. On the contrary, these
multiple analyses favor in many cases an integrate view of the
relevance of a given protein complex both in vitro and in the cell.
For example, protein interaction measurements in the cellular context provide a characterization of the interactions in the presence of
potential competitors or of post-translational modifications at different steps of the cell cycle. Biophysical approaches nicely complement these data by confirming or not the direct interaction with
purified proteins and ligands. Biophysical measurements are central
to characterize the strength, the specificity, and the stoichiometry
of the interactions in a quantitative manner. Interactions measured
in cells and not confirmed in vitro can also be informative of the
role of an additional partner or the need for post-translational
modifications eventually absent on purified proteins used for in vitro
studies.
A large panel of biophysical approaches are used nowadays to
perform quality controls and interaction analyses on macromolecular samples (some of these are presented in other chapters of this
issue). Each methodology offers complementary information and
comes with specific requirements on sample preparation. We present here protocols for Isothermal Titration Calorimetry (ITC) and
MicroScale Thermophoresis (MST) analyses of the interactions
between a protein complex and DNA substrates. The complex is
formed by the heterodimer Ku70–Ku80 (or Ku). Ku is a core factor
of the main DNA Double-Strand Break (DSB) repair pathway in
human, called Non-Homologous End-Joining (NHEJ). Ku recognizes the DSB ends through its ring-shaped structure [3]. The
heterodimer has a high affinity, in the nanomolar range, for
double-strand DNA (15 bp minimum). The K d of the Ku–DNA
interaction was initially characterized by gel shift assays (EMSA) [4]
and fluorescent anisotropy analyses [5]. Ku binds DNA in a
sequence-independent manner and in vitro one Ku molecule can
thread on DNA with one Ku bound every 15–20 bp of DNA
duplex [3, 6]. Interestingly, a super resolution microscopy study
showed that in cells only one or two Ku molecules bind to DSB
ends, suggesting a limited threading of Ku from the DNA DSB
ends in a cellular context [7]. Ku plays also a central role for the
recruitment of the enzymatic activities (nucleases, polymerases,
ligases) that process and ligate the DSB ends [8, 9]. Our laboratory
recently described at the molecular level the mechanism of recruitment by Ku of some of the downstream NHEJ factors [10]. Here,
we present the measurements by ITC of the thermodynamic parameters of the interaction between Ku and DNA substrates, and we
126
Amandine Gontier et al.