Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining

•Artemis nuclease is involved in the processing of DSB subsets by C-NHEJ and probably HRR.•We study here for the first time the role of Artemis nuclease in A-EJ.•DSB repair is analyzed in wild-type and Artemis deficient NALM-6 lymphocytes.•DSB repair is analyzed in normal and Artemis deficient human fibroblasts.•Our results validate the role for Artemis in C-NHEJ and HRR and show a role in A-EJ. Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis−/−, DNA ligase 4−/− (LIG4−/−), and LIG4−/−/Artemis−/− double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways.