NHEJ, DNA binding, and degradation of Ku80 using Ku-depleted (ΔKu) extract complemented with full-length or truncated Ku80 and Ku70

After mock or Ku depletion and RNase treatment, mRNAs encoding Ku70 and either full-length Ku80, Ku80 (ΔC), or Ku80 (ΔNΔC) were translated in the extract. S-methionine was added as indicated to radioactively label the translated proteins. (A) Extracts were immunoblotted with antibodies against Ku80 and Ku70. C-terminally truncated Ku80 cannot be detected with the anti-Ku80 antibody. (B) Autoradiograph of extracts labeled with S-methionine visualizing the C-terminally truncated Ku80 as well full-length Ku80 and Ku70. (C) NHEJ assay. Xmn1-digested pBluescript SK+ was added to mock- or Ku-depleted extracts after translation. At time points indicated, samples were removed, and DNA was purified by proteinase K digestion and phenol extraction. The resulting Southern blot was probed with pBluescript SK+ and exposed to film. Lanes 1, mock-depleted extract; lanes 2, ΔKu extract; lanes 3, ΔKu extract + Ku80 + Ku70; lanes 4, ΔKu extract + Ku80 ΔC + Ku70; lanes 5, ΔKu extract + Ku80 ΔNΔC + Ku70. s, linear substrate; c, internal 1-kb control; u, uncut plasmid; sm, supercoiled monomer; lm, linear monomer; nm/sd, nicked monomer/supercoiled dimer; ld, linear dimer; m, higher order multimers. (D) Autoradiograph of labeled proteins copurified with SB-DNA beads, revealing the binding of Ku80 truncations to SB-DNA beads and their modifications. (E) Degradation assay. Xmn1-digested pBluescript SK+ was added to extracts after translation in the presence of S-methionine. Autoradiograph of extract samples taken at the indicated time points is shown. (F) Quantification of results in E, using a phosphorimager. Left, loss of Ku80 (unmodified band); right, Ku70.Copyright information:Taken from "Ku80 removal from DNA through double strand break–induced ubiquitylation"The Journal of Cell Biology 2008;182(3):467-479.Published online 11 Aug 2008PMCID:PMC2500133.