Article Figures & Data
Figures
- FIG. 1.
The Ku80 carboxy terminus is important for DSB repair but not for recruitment of Ku70/80 to DSBs. (A) Schematic overview of the Ku80 deletion mutants used in the present study. (Top panel) The Ku80-WT (wild type) protein consists of 732 amino acids. (Middle panel) The Ku80-718 mutant lacks 14 carboxy-terminal amino acids. (Bottom panel) The Ku80-598 mutant lacks the entire carboxy terminus. In the present study we alternate between the use of a 598 and a 569 mutant. (B) IR survival of Xrs6 cells and (YFP-labeled) Ku80-WT-, Ku80-569-, or Ku80-718-complemented Xrs6 cells. (C) Purified Ku70/Ku80-WT and Ku70/Ku80-598 dimers. Proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue. (D) EMSA analysis, demonstrating the capability of the Ku70/Ku80-WT and Ku70/Ku80-598 dimers to associate with DNA in vitro. With decreasing amounts of unlabeled competitor DNA, the relative amount of detectable Ku-DNA species increases. (E) Accumulation of (YFP-labeled) Ku80-WT, Ku80-569, and Ku80-718 at DSB sites created by 365-nm laser IR in vivo. The white circle at t = 0 indicates the irradiated spot.
- FIG. 2.
The Ku80 carboxy terminus is not required for XRCC4/ligase IV or DNA-PKCS recruitment to DSBs. (A) EMSA analysis, demonstrating the ability of Ku70/Ku80-WT and Ku70/Ku80-598 to form DNA-bound complexes with XRCC4/ligase IV. (B) EMSA analysis, demonstrating the ability of Ku70/Ku80-WT and Ku70/Ku80-598 to form DNA-bound complexes with increasing concentrations of DNA-PKCS (30, 100, and 300 ng per reaction). (C) In vivo accumulation of YFP-DNA-PKCS at laser-induced DSB sites in Ku80-WT (V3) or Ku80-569 (complemented Xrs6) cells. The white circle at t = 0 indicates the irradiated spot. (D) FRAP analysis of YFP-DNA-PKCS at DSB sites in V3 (Ku80 competent CHO cells)- or Ku80-569-complemented Xrs6 (Ku80-deficient CHO cells).
- FIG. 3.
The Ku80 carboxy terminus is not essential for DNA-PKCS kinase activity. (A) In vitro DNA-PKCS kinase activity in the presence of Ku70/Ku80-WT or Ku70/Ku80-598. Kinase activity is determined by measuring phosphorus incorporation into a p53-based peptide (A) or into the DNA-PKCS band (B). (C) In vitro autophosphorylation of DNA-PKCS residues 2056, 260, and 2647 in the presence of Ku70/Ku80-WT or Ku70/Ku80-598. (D) In vivo phosphorylation of the DNA-PKCS Ser2056 residue at DSB sites in V3 (Ku80-competent CHO cells) or Ku80-569-complemented Xrs6 (Ku80-deficient CHO cells). The phospho-specific signal colocalizes with YFP-DNA-PKCS.
- FIG. 4.
Deletion of the Ku80 carboxy terminus alters end joining and V(D)J recombination characteristics. (A) Schematic depiction of the end-joining assay. Only the use of microhomology-mediated joining results in the formation of a BstXI site. The relative contribution of microhomology is estimated by analysis of the BstXI sensitivity of the joints. (B) End-joining assay in Xrs6 cells and (YFP-labeled) Ku80-WT-, Ku80-569-, or Ku80-718-complemented Xrs6 cells. Junctions were PCR amplified, and PCR products were digested with BstXI. (C) Schematic depiction of the V(D)J recombination assay. The formation of coding joints without loss of sequence or on a 4-bp microhomology results in the formation of an NgoMI or a NotI site, respectively. Only precise signal joints contain an ApaLI site. (D) V(D)J recombination assay, analyzing the formation and sequence of both coding joints and signal joints in Xrs6 cells and (YFP-labeled) Ku80-WT-, Ku80-569-, or Ku80-718-complemented Xrs6 cells.
- FIG. 5.
The Ku80 carboxy terminus stimulates Artemis-mediated DNA end processing. (A) In vitro hairpin processing assay. In the left panel, a hairpin opening requires the presence of both Artemis and Ku70/80. In the right panel, the hairpin opening activity is significantly reduced when Ku70/Ku80-598 is used instead of Ku70/Ku80-WT. Equal amounts of Ku70/Ku80-598 and Ku70/Ku80-WT were used. (B) Titration of Ku70/Ku80-WT in the hairpin opening assay. Even when Ku70/Ku80-WT is used at a 10-fold-lower concentration than Ku70/Ku80-598, the hairpin opening is still markedly more prevalent in the Ku70/Ku80-WT reaction. From left to right, the Ku70/Ku80-WT concentrations were 10, 5, 2.5, and 1 ng/μl, while Ku70/Ku80-598 was present at 10 ng/μl.
- FIG. 6.
Different roles for the Ku80 domains. This model proposes that the amino terminus and/or the central region of Ku80 are necessary to effectively recruit and activate DNA-PKCS. The Ku80 carboxy terminus stimulates autophosphorylation of the Thr2609 and possibly the Thr 2647 residues of DNA-PKCS, as well as activation of the Artemis endonuclease.
