ATR Homolog Mec1 Is Differentially Regulated at Different Times of DNA Damage Dependent Cell Cycle Arrest

Abstract

Budding yeast S. cerevisiae activates DNA damage checkpoint (DDC) in response to a single irreparable double strand break (DSB) prior to anaphase. One of the most upstream events in DDC signaling is the activation of PI(3) kinase-like kinase (PIKK) Mec1 (ATR in humans). Upon its activation, Mec1 activates downstream targets in the DDC pathway to assure proper cell cycle arrest. If the DSB irreparable, the DDC signaling is turned off after 12 to 15 hours to release the cells from this Mec1-dependent arrest. How cells determine the length of cell cycle arrest is not fully understood. We hypothesized that Mec1 could be regulated differentially during cell cycle arrest to track the length of the arrest. To test this, we mutated 3 possible PIKK target sites on Mec1; S38Q to S38A and S38E, T1902Q to T1902A and T1902E, T1964Q to T1964A and T1964E. Our data indicate that T1902E, S1964E and T1902E+S1964E are sensitive to MMS, UV, zeocin, phleomycin and HU. S38A is also sensitive to phleomycin and zeocin. Moreover, S38A and T1902A+S1964A fail to adapt after the induction of a single irreparable DSB, whereas T1902E+S1964E fails to arrest the cell cycle due to DNA damage. In addition, we assayed the phosphorylation levels of downstream checkpoint effector kinase Rad53 with Western blotting as a proxy for DDC activity. We observed that T1902E+S1964E mutant has no detectable Rad53 hyperphosphorylation, while adaptation-defective mutants S38A and T1902A+S1964A have elevated levels of hyperphosphorylated Rad53 compared to wild-type cells. Taken together, these results indicate that Mec1 is differentially regulated at different times of DNA damage dependent cell cycle arrest through yet unknown mechanisms.