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. 2014 Dec 15;28(24):2693–2698. doi: 10.1101/gad.252478.114

Figure 2.

Figure 2.

The nuclease Artemis prevents DNA end resection and HR repair. (A) Loss of Artemis renders BRCA1-deficient cells resistant to PARP inhibition. HeLa cells were transfected with the indicated siRNAs and treated with 1 μM PARPis. Colony formation was quantified relative to colonies formed in untreated cells from the same setting. Data are represented as the mean ± SEM (n = 3). Knockdown efficiency of Artemis or BRCA1 was confirmed by Western blotting with the indicated antibodies. (B) Artemis suppresses RAD51-dependent HR repair. HeLa cells were transfected with the indicated siRNAs. At 3 h post-irradiation (10 Gy), cells were processed for RAD51 immunofluorescence. RAD51 foci were quantified (at least 400 cells were counted for each cell). Data are represented as the mean ± SEM (n = 3). (C) Artemis acts to prevent end resection. HeLa cells were transfected with the indicated siRNAs. At 3 h post-irradiation (10 Gy), cells were processed for RPA immunofluorescence. Shown is the quantitation of RPA foci-positive cells (at least 400 cells were counted for each cell). Data are represented as the mean ± SEM (n = 3). (D) Artemis knockdown reduces radial chromosomes in BRCA1-deficient cells. HeLa cells were transfected with the indicated siRNAs and treated with 1 μM PARPis. Metaphases were analyzed for radial chromosomes (n = 50 metaphases analyzed in each case). Data are represented as the mean ± SEM (n = 3). (E) The nuclease activity of Artemis is required for the prevention of end resection and HR repair. HeLa cells were reconstituted with empty vector, siRNA-resistant Artemis wild-type, or Artemis nuclease-inactive mutant (H35AD37N). Next, HeLa cells as well as these reconstituted cells were transfected with the indicated siRNAs and treated with 1 μM PARPis. Colony formation was quantified relative to colonies formed in untreated cells from the same setting. (Left panel) Data are represented as the mean ± SEM (n = 3). (Top panel) Knockdown efficiency of Artemis or BRCA1 was confirmed by Western blotting with the indicated antibodies. SFB empty vector (Mock), SFB-tagged wild-type Artemis, and H35AD37N mutant Artemis were transfected into 293T cells, and Artemis complexes were first pulled down by streptavidin beads, then eluted by biotin buffer, and pulled down again by S protein beads. (Bottom panel) In vitro nuclease assays of wild-type and H35AD37N mutant Artemis were performed as described in the Materials and Methods.

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