Artemis is a member of the SNM1/PSO2 gene family, the archetypical member of which was identified in budding yeast (Saccharomyces cerevisiae) as a factor required for efficient DNA interstrand cross-link repair (23, 53). Members of this family, which in humans also include SNM1, SNM1B, ELAC2, and CPSF73 (15, 27, 57), share a region of homology termed the SNM1 domain, which contains a metallo-β-lactamase fold and an appended β-CASP (for metallo-β-lactamase-associated CPSF Artemis SNM1/PSO2) domain that is a predicted nucleic acid binding motif (7, 41). Outside the SNM1 domain, the sequences of the yeast and human proteins are different. The function of yeast Snm1 remains largely unresolved, although several studies have indicated that it is involved in repairing double-strand breaks (DSBs) resulting from processing of interstrand cross-links (31, 35, 40). Artemis was originally identified molecularly as deficient in a human radiosensitive severe combined immunodeficiency syndrome (RS-SCID) (41), which is characterized by a defect in V(D)J recombination resulting in premature arrest of both B- and T-cell maturation. In addition, patient cell lines exhibited greater sensitivity to ionizing radiation (IR) than normal cells (9, 42, 44). RS-SCID resembles murine SCID caused by defects in DNA-PK, a protein complex involved in both V(D)J recombination and repair of DSBs via the nonhomologous end-joining (NHEJ) pathway. These findings have also been confirmed in a mouse model in which the Artemis gene was disrupted by gene targeting (51). Biochemical studies of Artemis have shown that it possesses a 5′→3′ exonucleolytic activity on single-stranded DNA, and when complexed with DNA-PKcs, it acquires endonucleolytic activity on 5′ and 3′ overhangs and the ability to open DNA hairpins (34). This latter activity is consistent with the observed defect in coding joint formation in Artemis-deficient cells. The nuclease function of Artemis appears to reside in the conserved SNM1 domain. In addition, it was shown that Artemis is a substrate of the kinase activity of DNA-PKcs in vitro. DNA-PKcs is a member of a family of large phosphatidylinositol-3-OH kinase-like kinases (PIKKs) that includes the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and RAD3-related (ATR) gene products (reviewed in reference 16). These findings define a role for Artemis in V(D)J recombination, and with the hypersensitivity of Artemis-deficient cells to IR irradiation, also indicate a role for this gene in the cellular response to DNA damage. It has been proposed, although not formally demonstrated, that the radiosensitivity of Artemis-deficient cells is due to a defect in NHEJ (34). ATM and ATR are two central signaling kinases that mediate pleiotropic response to DNA damage, including activation of cell cycle checkpoints, DNA repair pathways, transcription, and apoptosis (reviewed in references 1, 16, 56, and 63). Although they have some functional redundancy, ATM and ATR have specialized roles that appear to operate in parallel in the response to DNA damage: ATM primarily responds to the induction of DSBs, while ATR is activated by many forms of DNA damage and replication inhibitors. Many downstream phosphorylation targets of ATM and/or ATR have been identified, including p53, BRCA1, Nbs1, Smc1, FANCD2, Chk1, Chk2, and Rad17. These proteins are mediators and transducers of the stress signal emanating from these two PIKKs. Some of these substrates, as well as ATM and ATR, are found together in a large multifactorial association of proteins referred to as the BRCA1-associated surveillance complex (BASC), which may function to recognize unusual or aberrant DNA structures and to activate DNA repair and checkpoint pathways (61). Thus, the BASC is thought to be a complex that both senses and transduces the DNA damage signal. In this report we demonstrate that Artemis-deficient cells are not, in fact, significantly defective in NHEJ following exposure to IR. Rather, Artemis is shown to interact with known checkpoint proteins and to be phosphorylated by ATM and ATR in vitro. In addition, Artemis is phosphorylated in vivo after exposure of cells to genotoxic stress: this modification is dependent upon both DNA-PK and ATM after IR and upon ATR in response to UV radiation. Further, we show that Artemis-deficient cells exposed to IR are defective in a G2/M DNA damage checkpoint. These findings define a novel function for Artemis as a checkpoint protein and suggest a new basis for the radiosensitivity and chromosomal instability of Artemis-deficient cells.