Abstract
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.
Highlights
DNA double-strand breaks (DSBs) are among the most genotoxic DNA lesions
The tags did not interfere with DNA-PKCS activation, and Enhanced Cyan Fluorescent Protein (ECFP)-Ku70 and enhanced yellow fluorescent protein (EYFP)-Ku80 were able to complement the genetic defect in Ku70 and Ku80 deficient cells, respectively ([9,17] and Figure S1D), showing that the tagged Ku70 and Ku80 proteins could be used for functional studies on Ku protein behavior
As changes in Förster resonance energy transfer (FRET) efficiency could be used to monitor Ku-DNA end binding in complex mixtures, we investigated the effect of DNA-PKCS on exchange kinetics from DNA ends using the FRET assay
Summary
DNA double-strand breaks (DSBs) are among the most genotoxic DNA lesions They can be induced by ionizing radiation or certain chemicals, but they are part of normal cellular processes, such as V(D)J recombination in lymphocytes. Correct repair of these breaks is very important to prevent chromosomal deletions and translocations, which can cause cancer or cell death [1]. Once bound to DNA, Ku70/80 attracts the catalytic subunit of the DNA-dependent protein kinase (DNA-PKCS) This interaction is mediated by the Ku80 C-terminus and other contact points between the Ku heterodimer and DNA-PKCS [6,7,8,9,10,11,12]. The final step in the NHEJ reaction is ligation of the juxtaposed DNA ends by the ligase IV/XRCC4 complex, which requires the XLF/Cernunnos protein and/or PAXX proteins [16]
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