Artemis Is a Phosphorylation Target of ATM and ATR and Is Involved in the G₂/M DNA Damage Checkpoint Response

Abstract

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.

We thank the following investigators for gifts of reagents. The ATR and ATM expression constructs were provided by Karlene Cimprich and Michael Kastan, respectively. P11 and P14 cell lines were provided by J. P. de Villartay. The ATR^flox/− cells were provided by Stephen Elledge, and the RAD17^flox/− cells were provided by Lei Li. We thank Lei Li for comments on the manuscript.

This work was supported in part by grants from the NIH (CA52461, CA96574, CA90270, and ES07784).