The ARF-GAP Gcs1 is required for adaptation to the DNA damage checkpoint.
Jacob C. Harrison (1), Ayelet Arbel-Eden (1), Chiara Lucca (2), Marco Foiani (2), James E. Haber (1)
(1) Rosenstiel Center, Brandeis University, 415 South St, Waltham, MA, 02454, USA;
(2) Universita degli Studi di Milano and Instituto FIRC di Oncologia Molecolare, Milan 20133 Italy
Cells suffering a single unrepairable double-strand break (DSB) activate the DNA damage checkpoint and exhibit a long but transient arrest prior to anaphase. After 8-12 hours cells re-enter the cell cycle in a process termed adaptation. Adaptation appears to down-regulate the Mec1/Ddc2 complex, but the molecular events triggering and executing adaptation are unknown. Here we show that the ARF-GAP Gcs1 is required for adaptation. Cells lacking Gcs1 are also sensitive to DNA damage caused by hydroxyurea and MMS. None of the other ARF-GAP mutants show an adaptation defect or DNA damage sensitivity, but overexpression of any of them (AGE1, AGE2, GLO3, SPS18) suppresses both defects in gcs1 mutant cells. This result, as well as the partial adaptation defect of arf1 and arf2 mutants, indicates that Gcs1's regulation of ARF is required for adaptation. All other adaptation-defective mutants tested maintain Rad53 hyperphosphorylation even at 24 hr. Cells lacking Gcs1, in contrast, remain arrested despite apparently normal dephosphorylation of Rad53 during the arrest. This result places Gcs1 further downstream than Rad53 and the other proteins known to function in adaptation. Interestingly, deletion of the spindle checkpoint protein Mad2 partially suppresses the adaptation defect of gcs1 mutant cells. This result is consistent with a more downstream role for Gcs1 and is further support for a role for Mad2 in some aspects of the DNA damage checkpoint.
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