Caught in the Middle: A Rigid DNA Label That Provides an Incisive Picture of DNA Conformational Flexibility in Protein–DNA Complexes

Measurement of the conformation of DNA in protein-DNA complexes is important to decipher the role of the DNA conformation and dynamics in protein recognition and function. In this work, we report a rigid nucleotide-independent spin label that places paramagnetic Cu(II) within the DNA helix. The labeling strategy exploits the chelation of Cu(II) to two 8-aminoquinoline moieties, one in each strand. Because the rigidity of the probe avoids potentially confounding motions of the label itself, EPR signals can resolve 2-3 Å changes in interspin distance; the breadth of the distance distribution reports dynamic fluctuations from the most probable conformation. Continuous wave and pulsed electron paramagnetic resonance spectroscopy (EPR) shows that Cu(II) coordinates properly to the labeling sites. Measurements of the interspin dipolar interaction on DNA oligonucleotides with two labels placed at various distances demonstrate that the label provides accurate and narrow distance distributions sensitive to DNA conformation and flexibility. Molecular dynamics simulations support these interpretations. We utilized this label to measure the conformations of DNA when the type II restriction endonuclease <i>Eco</i>RV binds to its specific recognition sequence. The results provide in-solution evidence that the <i>Eco</i>RV endonuclease induces axial DNA bending in the absence of metal ions, contrary to a long-standing belief. Furthermore, the distance distribution narrows upon protein binding and even further on subsequent metal binding, implying that bound protein constrains the bending dynamics of DNA. This method provides a novel and accurate approach to assess DNA conformation and dynamics in solution.