Involvement of Brca1 in S-Phase and G₂-Phase Checkpoints after Ionizing Irradiation

Abstract

Cell cycle arrests in the G₁, S, and G₂phases occur in mammalian cells after ionizing irradiation and appear to protect cells from permanent genetic damage and transformation. Though Brca1 clearly participates in cellular responses to ionizing radiation (IR), conflicting conclusions have been drawn about whether Brca1 plays a direct role in cell cycle checkpoints. Normal Nbs1 function is required for the IR-induced S-phase checkpoint, but whether Nbs1 has a definitive role in the G₂/M checkpoint has not been established. Here we show that Atm and Brca1 are required for both the S-phase and G₂ arrests induced by ionizing irradiation while Nbs1 is required only for the S-phase arrest. We also found that mutation of serine 1423 in Brca1, a target for phosphorylation by Atm, abolished the ability of Brca1 to mediate the G₂/M checkpoint but did not affect its S-phase function. These results clarify the checkpoint roles for each of these three gene products, demonstrate that control of cell cycle arrests must now be included among the important functions of Brca1 in cellular responses to DNA damage, and suggest that Atm phosphorylation of Brca1 is required for the G₂/M checkpoint.

ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistance of Jim Houston, Angela Justman, and Diane Woods. We thank all members of the Kastan laboratory for helpful discussions, Yossi Shiloh for providing complemented A-T cells, and David Livingston for providing the wild-type BRCA1 cDNA.

This work was supported by grants from the National Institutes of Health (CA71387 and CA21765) and by the American Lebanese Syrian Associated Charities of the St. Jude Children's Research Hospital.