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Chair(s): Chen, David; Powell, Simon
(MS001) ATM-dependent double-strand break repair and checkpoint functions co-operate to maintain chromosomal stability after ionizing irradiation.
Krempler, Andrea1, Deckbar, Dorothee1, Tchouandong, Leopoldine1, Walker, Sarah2, Jeggo, Penny2, Lobrich, Markus*,1, 1 Biophysics Department, Homburg, Saar, Germany2 Genome Damage and Stability Centre, East Sussex, BN, UK
ABSTRACT- Unrepaired double-strand breaks (DSBs) constitute a major threat for proliferating cells. The kinase ATM, mutated in ataxia telangiectasia (A-T) cells, is a key protein in the signalling cascades that initiate checkpoints after ionizing radiation (IR). A-T cells are impaired in their ability to induce these checkpoints and are characterized by pronounced radiosensitivity. Recently, it has been demonstrated that ATM has an additional, direct role in the repair of a subset of DSBs which contributes to the radiosensitivity of A-T cells. This ATM-dependent repair pathway includes the nuclease Artemis, and Artemis-deficient cells show the same radiosensitivity as A-T cells. Using flow cytometry analysis, we investigated the passage of BrdU-labelled cells through the cell cycle. Compared with wild-type cells, both ATM- and Artemis-deficient cells showed a longer accumulation in the G2 phase, and the duration of the arrest correlated with dose. Investigation of the G2/M checkpoint by histone H3 analysis demonstrated that Artemis-deficient cells are capable to induce this checkpoint, whereas ATM-deficient G2 cells continued to enter mitosis. After higher doses of IR, Artemis cells sustained this checkpoint even longer than wild-type cells. To investigate this in more detail, we used g-H2AX staining in combination with a G2-specific marker protein to visualize DSBs in G2 cells. Quantitative analysis revealed that the repair defect of A-T and Artemis cells, initially described in non-cycling G0 cells, is also observed in G2 phase. Interestingly, chromosomal studies in colcemide-blocked metaphases from ATM- and Artemis-deficient cells showed a difference in the amount of chromosome breaks. The break number in Artemis cells that had transversed the checkpoint was lower than in A-T cells, but higher than in wild-type cells. These studies provide direct evidence that ATM-dependent DSB repair and checkpoint functions cooperate to prevent cells from entering the next cell cycle phase with an excess of DSBs.
Key words: ataxia telangiectasia, double strand break repair, ionizing radiation, checkpoint control
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