UC San Diego

Faithful genome duplication is fundamentally important for all organisms. When cells encounter DNA replication stress, from difficulties in replicating either repeated sequence or aberrant DNA structure resulting from UV radiation or genotoxic chemicals, replication forks are stalled. This impediment may result in the collapse of replication forks and consequently DNA damage. Eukaryotes have evolved a signaling pathway, namely DNA replication checkpoint, which regulates cellular responses for cell viability and maintenance of genome stability under replication stress. Defects in replication checkpoint results in genomic instability, cell death and cancer. Activation of effector kinases is the hallmark of DNA replication checkpoint. They are responsible for coordinating many cellular responses after replication stress, ranging from the stabilization of stalled replication forks to the cell cycle arrest. While the functions of the effector kinases have long been appreciated, the molecular mechanisms for their activation remain elusive. Studies presented here, which utilize the biochemical and genetic amenability of budding yeast, Saccharomyces cerevisiae, reveal the molecular basis of effector kinases activation. Discoveries in Chapter 2 showed the reconstitution of the activation and phosphorylation state of an effector kinase, Dun1. Analysis on its phosphorylation reveals the specific phosphorylation on its activation loop that triggers it activation. Additionally, this specific residue at the activation loop of all Chk2 family kinases are conserved. We thus suggested this activation loop phosphorylation to be a conserved regulation for the activation of Chk2 family kinases, including Dun1 and Rad53 in budding yeast. Mrc1, as part of replication machinery, serves as a docking site at stalled replication fork for the recruitment of another effector kinase Rad53. Mec1, a PIKK kinase, is the upstream replication checkpoint sensor. Both Mec1 and Mrc1 have been implicated to be crucial for Rad53 activation. Based on the studies in Chapter2, a biochemical approach using the reconstitution of Rad53 activation has led to the discovery of efficient Rad53 activation by Mec1 mediated through Mrc1. Analysis on the reconstituted system showed that instead of a two-step linear pathway, the primary role of Mrc1 is to promote Rad53 phosphorylation by Mec1 by catalyzing the enzyme- substrate interaction between Mec1 and Rad53