Dephosphorylation of the Cdc2 kinase by the Cdc25 tyrosine phosphatase is the universally conserved trigger for mitotic entry. Cdc25 is also the point of convergence for checkpoint signaling pathways which monitor the genome for damaged DNA and incomplete replication. In addition, Cdc25 is inhibited by a MAP kinase cascade in the event of osmotic, oxidative and/or heat stress. These pathways inhibit cell cycle progression by phosphorylating Cdc25 resulting in its association with 14-3-3 and nuclear export. Although Cdc25 can be observed leaving the nucleus following inhibitory signals it is controversial whether phosphorylation, 14-3-3 binding or export itself is required for checkpoint proficiency. In fission yeast, Cdc25 is phosphorylated in vitro on 12 serine and threonine residues by the effector kinase of the DNA replication checkpoint, Cds1. Nine of these residues reside in the N-terminal regulatory region, while three are found in the extreme C-terminus of the protein. We show here that phosphorylation the nine N-terminal residues, nor any of the 12 in vitro sites, are required for enforcement of the DNA replication checkpoint. In lieu of Cdc25 phosphorylation the phosphatase is rapidly degraded and mitotic entry prevented by the action of the Mik1 kinase, targeting Cdc2. Thus, multiple mechanisms exist for preventing mitotic entry when S-phase progression is inhibited. The three C-terminal in vitro phosphorylation sites have not previously been examined in fission yeast. However, homology exists between the S. pombe protein and the Cdc25 orthologues in humans, Xenopus and Drosophila in this region. We report here that in S. pombe these sites are required to prevent mitotic entry following replication arrest in the absence of Mik1, and in the maintenance, but not establishment, of arrest following DNA damage. Our previous work showed that Cdc25 nuclear import requires the Sal3 importin-β but at the time we were unable to show a direct interaction between these two proteins. The final chapter of this thesis proves physical interaction by co-immunoprecipitation. Cdc25 mutants lacking all twelve putative Cds1 sites show nuclear localization during mitosis in a sal3- background, effectively reversing the cell cycle regulated pattern of accumulation of the phosphatase. / Thesis (Ph.D, Biology) -- Queen's University, 2011-06-20 12:16:15.71

<p>All living things must replicate their DNA. Despite being essential for life, this process is also inherently dangerous. Replication stress, which induces replication fork stalling, is an unavoidable risk that can trigger potentially harmful changes to the genome. Eukaryotes have a replication checkpoint pathway that stabilizes stalled replication forks to prevent damage. One of the critical protein interactions in this pathway, between Dbf4 and Rad53, pauses the cell cycle in budding yeast. This is important to give the cell time to recover from stress. The molecular details of this interaction were investigated to shed light on how this association is regulated by the cell. The structure of an N-terminal domain from Dbf4 was solved through X-ray crystallography and discovered to have a modified <em>BR</em>CA-1 <em>C</em>-<em>t</em>erminal (BRCT) fold, which included an additional N-terminal helix. This domain could interact with the <em>F</em>ork<em>H</em>ead <em>A</em>ssociated 1 (FHA1) domain from Rad53 <em>in vitro</em>, and the additional helix was necessary for complex formation. Although the FHA1 domain has a well-characterized binding site for phospho-epitopes, a combination of chemical cross-linking and NMR spectroscopy experiments demonstrated that the N-terminal domain from Dbf4 is contacting an alternative surface. However, the full-length Dbf4 protein <em>in vivo</em> may be contacting both this distal site and the phospho-epitope binding pocket. This bipartite interaction between Dbf4 and Rad53 would lend specificity to the complex and also suggests a kinase may be regulating the association. As FHA and BRCT domains are prevalent in eukaryotic nuclear proteins, these findings are instructive for how these domains mediate interactions in other signaling pathways.</p> / Doctor of Philosophy (PhD)

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