Mrc1 phosphorylation in response to DNA replication stress is required for Mec1 accumulation at the stalled fork

The D NA d amage r esponse (DDR) is a highly conserved signal transduction network that coordinates interconnecting pathways involved in the detection and repair of DNA lesions. The DNA replication stress response is a branch of the DDR involved in sensing replication stress, transducing the signal, and effecting a cellular response. This dissertation focuses on Mrc1 ( m ediator of r eplication c heckpoint), a central replication stress response signaling protein in the budding yeast Saccharomyces cerevisiae . It also highlights a global, in vivo screen-based approach to create a database of DDR-regulated phospho-peptides. Mrc1 is a mediator protein with roles in both DNA replication and signal transduction. In response to replication stress induced by hydroxyurea, Mrc1 forms a stable pausing complex at stalled forks and is phosphorylated by the Mec1 sensor kinase. Mrc1 then mediates the stress signal to activate the Rad53 effector kinase. mrc1 AQ mutants, which cannot be phosphorylated, are unable to mediate Rad53 activation. A structure-function analysis carried out to identify key functional domains revealed a central core region important for Mrc1's ability to promote efficient replication and mediate the stress signal. Surprisingly, it does not significantly affect Mec1 maintenance at the stalled fork. mrc1 AQ and mrc1Δ mutants, however, have profoundly reduced Mec1 accumulation, independent of Rad53 activity. This suggests that Mec1-dependent phosphorylation of Mrc1 is crucial for the establishment of a positive-feedback loop that promotes Mec1 accumulation at the stalled fork. These results provide insight into how Mrc1 mediates the DNA replication stress signal in S phase. In order to understand the kinase-regulated signal transduction pathways that comprise the DDR at the systems level, a thorough knowledge of kinase substrates is required. A screen in S. cerevisiae utilizing SILAC ( s table i sotope l abeling of a mino acids in c ulture) and mass spectrometry was successful in generating a vast database of DDR-regulated and kinase-dependent phospho-peptides. Additionally, we identified genes that control the inositide "metabolome" as a prospective DDR-regulated network. These results, along with the potential to identify other novel DDR-regulated proteins and networks, demonstrate the power of the screen to reveal a more comprehensive, global view of the DDR and its conserved mechanisms in higher eukaryotes.