Phosphorylation events surrounding the DNA damage response in 'Saccharomyces cerevisiae'

Protein phosphorylation mediated by checkpoint kinases is crucial for the cellular response to DNA damage. The sensor kinases Mec1 and Tel1 initiate the checkpoint signaling cascade by directly activating the checkpoint effector kinase Rad53. This checkpoint pathway, however, is responsive to normal endogenous replication as well. As a result an S-phase specific threshold for Rad53 activation exists, which allows cells to tolerate endogenous damage-like structures. Here we show that Rad53 itself is phosphorylated in a cell-cycle dependent manner independent of DNA damage signaling (Chapter 2). We propose that this is part of the cell-cycle regulated sensitivity of Rad53 to activation. This phosphorylation occurs in G2/M, persists until S phase onset and depends on both the polo-like kinase, Cdc5, and the cyclin-dependent kinase, Cdc28. These cell-cycle dependent phosphorylation events are located in the C-terminal part of Rad53. Serines 774 and 789 were shown to be phosphorylated by mass spectrometry. Mutation of these sites eliminated the cell-cycle dependent phosphorylation of Rad53 and partially impaired the activation of Rad53 in response to minor amounts of DNA damage in G2/M. This led to more rapid checkpoint adaptation in response to irreparable DNA damage. Thus, cell-cycle dependent phosphorylation in the C-terminal part of Rad53 enhances Rad53 activation in response to DNA double strand breaks (DSBs). Mec1 and Tel1 initiate a response to DNA damage independently of Rad53. The phosphorylation of histone H2A at serine 129 (γH2A) at DSBs by Mec1 and Tel1 has an important role in mediating DNA repair. This study shows that the occurrence of γH2A is not limited to DSBs, but also occurs at stalled replication forks (Chapter 3). Using chromatin immunoprecipitation high γH2A levels were monitored at hydroxyurea-stalled replication forks and depended nearly exclusively upon Mec1 kinase activity. Furthermore our study showed that γH2A not only occurs at damaged chromatin but in regions of normally replicating ...