Zhang, X. et al. Artemis is a phosphorylation target of ATM and ATR and is involved in the G2/M DNA damage checkpoint response. Mol. Cell. Biol. 24, 9207-9220

Department of Molecular Genetics, The University of Texas M D Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 11/2004; 24(20):9207-20. DOI: 10.1128/MCB.24.20.9207-9220.2004
Source: PubMed


Mutations in Artemis in both humans and mice result in severe combined immunodeficiency due to a defect in V(D)J recombination.
In addition, Artemis mutants are radiosensitive and chromosomally unstable, which has been attributed to a defect in nonhomologous
end joining (NHEJ). We show here, however, that Artemis-depleted cell extracts are not defective in NHEJ and that Artemis-deficient
cells have normal repair kinetics of double-strand breaks after exposure to ionizing radiation (IR). Artemis is shown, however,
to interact with known cell cycle checkpoint proteins and to be a phosphorylation target of the checkpoint kinase ATM or ATR
after exposure of cells to IR or UV irradiation, respectively. Consistent with these findings, our results also show that
Artemis is required for the maintenance of a normal DNA damage-induced G2/M cell cycle arrest. Artemis does not appear, however, to act either upstream or downstream of checkpoint kinase Chk1 or
Chk2. These results define Artemis as having a checkpoint function and suggest that the radiosensitivity and chromosomal instability
of Artemis-deficient cells may be due to defects in cell cycle responses after DNA damage.

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    • "Several reports suggest that Artemis is phosphorylated by ATM as well as DNA-PKcs [42], [45], [46]. Löbrich and Jeggo [47] reported that ATM is required for Artemis function in response to ionizing radiation, but not in V(D)J recombination. "
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    ABSTRACT: Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.
    PLoS ONE 08/2013; 8(8):e72253. DOI:10.1371/journal.pone.0072253 · 3.23 Impact Factor
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    • "In contrast to ATM, Artemis appears not to be implicated in direct checkpoint activation (5). However, it was suggested that ATM/ATR-catalyzed phosphorylation of Artemis facilitates the recovery from G2-block through regulation of CyclinB/Cdk1 activation (14,15). Biochemically, the Artemis protein is an exo- and endonuclease critically involved in the resolution of hairpin DNA structures (16), which are regular intermediates of V(D)J recombination. "
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    ABSTRACT: Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.
    Nucleic Acids Research 06/2012; 40(17):8336-47. DOI:10.1093/nar/gks604 · 9.11 Impact Factor
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    • "Inhibition of DNA-PK reveals that the radio-protective effect of Artemis over-expression was principally reliant on DNA-PK activity and to a lesser degree on ATM activity. In contrast, we find that Artemis phosphorylation, indicated by a mobility shift of Artemis protein, was primarily dependent on the activity of ATM, as has been reported previously [14,17,32]. Furthermore, in cells containing high levels of Artemis, cell proliferation following irradiation was more severely diminished by loss of DNA-PK activity than by ATM inhibition (compare Figure 6A & B). "
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    ABSTRACT: Background Artemis has a defined role in V(D)J recombination and has been implicated in the repair of radiation induced double-strand breaks. However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined. Our previous work suggests that Artemis is important for the repair of complex DNA damage like that inflicted by high Linear Energy Transfer (LET) radiation. To establish the contribution of Artemis in repairing DNA damage caused by various radiation qualities, we evaluated the effect of over-expressing Artemis on cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation. Results Our data reveal that Artemis over-expression confers marked radioprotection against both types of radiation, although the radioprotective effect was greater following high LET radiation. Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity. Together, these data suggest a DNA-PK dependent role for Artemis in the repair of complex DNA damage. Conclusions These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies.
    Radiation Oncology 06/2012; 7(1):96. DOI:10.1186/1748-717X-7-96 · 2.55 Impact Factor
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