Journal: The Journal of Cell Biology

Article Title: Ku80 removal from DNA through double strand break–induced ubiquitylation

doi: 10.1083/jcb.200802146

Figure Lengend Snippet: Ku80 bound to short oligonucleotide duplexes has less polyubiquitylation and is stable on DNA beads. (A) Steptavidin-coated beads bound to a biotinylated hairpin-containing 20-bp duplex DNA, a 49-bp double-stranded oligo containing a biotinylated 3′ end on one strand, or a 3,000-bp SB-DNA were incubated in extract for 30 min. Binding of Ku80, Cul1, and Skp1 were analyzed by immunoblot. (B) 20- and 49-bp oligo DNA beads and SB-DNA beads were used in the DNA release assay detailed in . (C) Quantification of B using a phosphorimager. Error bars denote the range of the data; n = 2.

Article Snippet: For DNA release assays, phosphorimager (Fujifilm) quantification of modified and unmodified Ku80 was normalized to an adjusted GFP-SBP signal for that lane.

Techniques: Incubation, Binding Assay, Western Blot, Release Assay

Journal: The Journal of Cell Biology

Article Title: Ku80 removal from DNA through double strand break–induced ubiquitylation

doi: 10.1083/jcb.200802146

Figure Lengend Snippet: DSBs lead to the proteasomal degradation of Ku80. (A) Cut linear DNA, supercoiled circular DNA, or buffer was added to extract containing 35 S-labeled Ku80. After incubation at 22°C for the indicated times, samples were taken. The asterisk indicates DSB-dependent modified Ku80. Radioactively labeled protein was visualized using a phosphorimager. (B) Quantification of the radioactivity of the entire lane in A using a phosphorimager ( n = 4). (C) Degradation, quantified as in B, of Ku80, Ku70, and Mre11 in the presence of cut DNA ( n = 3). (D) Degradation, quantified as in B, of Ku80 in the presence of cut DNA and buffer, 0.5 mg/ml wild-type ubiquitin, or 0.5 mg/ml ubiquitin-K48R ( n = 3). (E) Degradation of Ku80 in the presence of buffer, supercoiled DNA, or cut DNA. At indicated time points extract samples were spotted onto glass microfiber filters and precipitated in 10% TCA, and radioactivity was quantified using a liquid scintillation counter ( n = 4). (F) Degradation, quantified as in E, of Ku80 in the presence of either DMSO or 100 μM MG132 ( n = 4). Error bars denote one standard error of the mean.

Article Snippet: For DNA release assays, phosphorimager (Fujifilm) quantification of modified and unmodified Ku80 was normalized to an adjusted GFP-SBP signal for that lane.

Techniques: Labeling, Incubation, Modification, Radioactivity

Journal: The Journal of Cell Biology

Article Title: Ku80 removal from DNA through double strand break–induced ubiquitylation

doi: 10.1083/jcb.200802146

Figure Lengend Snippet: Nonfunctional Ku80 is ubiquitylated and degraded in response to DSBs. 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 1-543 (ΔC), or Ku80 183-543 (ΔNΔC) were translated in the extract. 35 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 35 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 35 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.

Article Snippet: For DNA release assays, phosphorimager (Fujifilm) quantification of modified and unmodified Ku80 was normalized to an adjusted GFP-SBP signal for that lane.

Techniques: Binding Assay, Autoradiography, Labeling, NHEJ Assay, Purification, Southern Blot, Plasmid Preparation, Degradation Assay

Journal: The Journal of Cell Biology

Article Title: Ku80 removal from DNA through double strand break–induced ubiquitylation

doi: 10.1083/jcb.200802146

Figure Lengend Snippet: N- and C-terminal Ku80 truncations are ubiquitylated and degraded in response to binding to DSBs. (A) Domain organization of Ku80 is modified from a previous review (for review see ). Ku80 domains include vWA, N-terminal von Willibrand A domain; Core, central core domain; CT, C-terminal region; PK, DNA-PKcs–interacting peptide . The core DNA-binding region is indicated in light blue, and the other regions of the protein included in the atomic structure are in yellow. Regions of the protein not included in the structure are white. Numbers to the left indicate amino acids included in the protein. An asterisk indicates the location of amino acid W275. Truncations were generated as indicated and labeled with 35 S in rabbit reticulocyte lysate. Proteins were then mixed with egg extract, to which was added either SB-DNA beads to assess DNA binding or cut linear DNA to measure degradation. Half-lives of the unmodified bands after addition of cut DNA, quantified using a phosphorimager, are indicated. (B) The structure of the human Ku70/Ku80 heterodimer bound to DNA . Residues up to the equivalent of amino acid 543 of the X. laevis Ku80 are included in the structure. Red, Ku70; yellow, Ku80; white, DNA. The region of Ku80 required for DNA binding ( X. laevis residues 244–540) is light blue. (C) Truncations were mixed with extract and incubated with SB-DNA beads for 45 min. Bound proteins were visualized by exposing to film. I, input; B, beads.

Article Snippet: For DNA release assays, phosphorimager (Fujifilm) quantification of modified and unmodified Ku80 was normalized to an adjusted GFP-SBP signal for that lane.

Techniques: Binding Assay, Modification, Generated, Labeling, Incubation

Journal: The Journal of Cell Biology

Article Title: Ku80 removal from DNA through double strand break–induced ubiquitylation

doi: 10.1083/jcb.200802146

Figure Lengend Snippet: Ku80-W275R is functional for NHEJ but is not released from DNA. (A) Radioactively labeled wild-type (WT) Ku80 or Ku80-W275R were mixed with egg extract and linear DNA was added. Samples were taken at the indicated time points, and degradation was quantified using a phosphorimager. Error bars denote the range of data; n = 2. (B) Wild-type Ku80 and Ku80-W275R were used in the release assay , and the results were visualized using a phosphorimager. (C) Quantification of results in B, including modified and unmodified Ku80. Error bars denote the range of data; n = 2. (D) Schematic of immunodepletion/translation experiment. Extract was immunodepleted using an anti-Ku80 antibody in the presence of RNase A, after which an RNase inhibitor was added along with tRNAs. mRNAs encoding Ku70 and either wild-type or mutant Ku80 were then added and allowed to translate in the extract. (E) Samples of extract after translation were immunoblotted and probed with antibodies against Ku70 and Ku80. For comparison, a dilution series of undepleted extract has been included. (F) After translation, Xmn1-digested pBluescript SK+ DNA was added to the extract. At indicated time points, samples were taken and treated with proteinase K and phenol/chloroform extraction. DNA was run on an agarose gel containing ethidium bromide, Southern blotted, probed with pBluescript DNA, and exposed to film. 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.

Article Snippet: For DNA release assays, phosphorimager (Fujifilm) quantification of modified and unmodified Ku80 was normalized to an adjusted GFP-SBP signal for that lane.

Techniques: Functional Assay, Labeling, Release Assay, Modification, Mutagenesis, Agarose Gel Electrophoresis, Plasmid Preparation