Fig 2: DNA-PKcs interaction is necessary for V(D)J recombination activities independent of autoinhibitory self-interaction. (A) Schematic of ARTEMIS indicating different protein domains (see Figure 3A for details). Below, full-length ARTEMIS proteins are represented as horizontal lines with x indicating mutations in the DNA-PKcs binding domain (K376V + R377V + R402P) and in the C-terminal autoinhibition domain (N456A + S457A + E458Q). On the right, DNA-PKcs and N:C self-interaction of ART-WT and mutants A–E are indicated by + and –, the results of the corresponding experiments are depicted in Figure 6B and Supplementary Figure S7. (B) Co-immunoprecipitation of full-length ARTEMIS proteins by the C-terminal ARTEMIS fragment ARM414-692, demonstrating loss of autoinhibitory self-interaction. HCT116 DCLRE1C–/– cells were transiently co-transfected with combinations of Myc-tagged ARM414-692 and V5-tagged full-length ARTEMIS, empty vector was used as negative control. The different interacting full-length proteins (circles and ellipses) and protein fragments (rectangles) are represented graphically at the top (not to scale). Immunological tags of IP (Myc) and Co-IP products (V5) are indicated in blue and green, respectively. Cell lysates were prepared and the Myc-tagged C-terminal fragments (ARM414-692) were immunoprecipitated (IP) with anti-Myc antibody. Western blotting was performed and co-immunoprecipitation (Co-IP) of V5-tagged full-length ARTEMIS (ART-WT/ARM) was compared to input using anti-V5 antibody. IP products were identified using anti-Myc antibody. Association of endogenous DNA-PKcs with the co-immunoprecipitated full-length ARTEMIS proteins was detected using anti-DNA-PKcs antibody. The positions of protein standards are given at the right in kDa. Anti-β-actin (ACTB) antibody was used as loading control in input lanes. Two independent co-immunoprecipitation experiments were performed, and comparable results obtained. (C) HCT116 DCLRE1C–/– cells were co-transfected with V(D)J recombination substrate (see Figure 5A), plasmids expressing RAG1, RAG2 and Myc-tagged full-length ARTEMIS of either wildtype sequence (WT) or containing mutations in the DNA-PKcs interaction domain or the C-terminal autoinhibitory domain as indicated below the columns. V(D)J recombination activity was determined as the percentage of recombination-positive cells out of the subpopulation of transfected cells minus negative control (see Supplemental Table S2). V(D)J recombination activities at 150 ng ART-WT/ARM expression plasmids are shown as means of three independent experiments with standard deviations. P values were calculated using a two-tailed t-test and are given at the top.

Fig 3: Amino acid exchanges in regions A and B of the DNA-PKcs interaction domain in ARTEMIS impair V(D)J recombination activity in HCT116 DCLRE1C–/– cells. HCT116 DCLRE1C–/– cells were co-transfected with V(D)J recombination substrate and plasmids expressing RAG1, RAG2 and Myc-tagged ARTEMIS proteins (wildtype or mutant). (A) Schematic of the V(D)J recombination substrate. Triangles indicate the Recombination Signal Sequences (RSS) surrounding the EGFP cDNA in antisense orientation. Arrows denote promoters driving expression of H2KK and EGFP. In the presence of transiently expressed RAG1, RAG2 and ARTEMIS proteins, the EGFP cassette is inverted, resulting in expression of EGFP protein. (B) V(D)J recombination activity was determined as the percentage of recombination-positive cells out of the subpopulation of transfected cells minus negative control (see Supplemental Table S1). V(D)J recombination activities at 150 ng ART-WT/ARM expression plasmids are shown as means of three independent experiments with standard deviations. P values for comparing ART-WT with each of the mutants were calculated using a two-tailed t-test and are given at the top. The amino acid exchanges in regions A and B of the DNA-PKcs interaction domain are specified below.