The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in Förster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5′ extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends.

Additional Metadata
Keywords DNA binding, DNA repair, DNA-PK, Non-homologous end-joining, Surface plasmon resonance (SPR)
Persistent URL dx.doi.org/10.3390/ijms21186725, hdl.handle.net/1765/130411
Journal International Journal of Molecular Sciences
Citation
Inagawa, T, Wennink, T. (Thomas), Lebbink, J.H.G, Keijzers, G, Florea, B.I, Verkaik, N.S, & van Gent, D.C. (2020). C-terminal extensions of ku70 and ku80 differentially influence dna end binding properties. International Journal of Molecular Sciences, 21(18), 1–12. doi:10.3390/ijms21186725