Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G₂/M Checkpoint Arrest

Abstract

ATM-dependent initiation of the radiation-induced G₂/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G₂ phase are repaired by DNA nonhomologous end joining (NHEJ), while ∼15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G₂/M checkpoint is maintained in irradiated G₂ cells, in light of our current understanding of G₂ phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1^−/− and MDC1^−/− cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank G. Smith for providing ATM inhibitor and E. Hoffman, A. Carr, and A. Lehmann for invaluable comments.

The M.L. laboratory is supported by the Deutsche Forschungsgemeinschaft (Lo 677/4-1/2), the Bundesministerium für Bildung und Forschung via Forschungszentrum Karlsruhe (02S8335 and 02S8355), and Forschungszentrum Jülich (03NUK001C). The P.A.J. laboratory is supported by the Medical Research Council, Association for International Cancer Research, Department of Health and EU grant (Integrated Project; DNA repair, LSHG-CT-2005-512113). Both laboratories are supported by EU grant FI6R-CT-2003-508842 (RiscRad).