Termination Of Replication Stress Signaling In Saccharomyces Cerevisiae

During DNA replication, replication forks are prone to stall and collapse. To prevent genomic instability, DNA damage checkpoint (DDC) kinases are activated and coordinate various cellular responses. In S. cerevisiae, the key DDC kinase Rad53 plays crucial roles in the regulation of transcription, dNTP levels, fork stability, cell cycle progression and origin firing. As part of the checkpoint response, Rad53 activation results in a major arrest in the cell cycle, which is thought to be important to increase the time in which DNA repair systems can function before the onset of mitosis, therefore preserving the faithful transmission of the genetic material to daughter cells. Although checkpoint signaling is beneficial for preventing genomic instability, Rad53 signaling needs to be turned off following repair to allow for resumption of cell proliferation. Similarly, in humans, activation of checkpoint kinases CHK1 and CHK2 must also be under stringent control to allow for cell proliferation. While different mechanisms for down-regulation have been identified, how they function together to efficiently control checkpoint signaling is not known. In particular, it is not clear how these mechanisms function locally at lesions or in diffused nuclear pools of Rad53 and how they are coordinated. Work from my thesis, summarized in the next paragraph, has elucidated important aspects of the spatiotemporal coordination of checkpoint down-regulation by multiple, distinct mechanisms. In yeast, several phosphatases have been implicated in regulating checkpoint deactivation. Recently, our group uncovered a phosphatase-independent mechanism for down-regulating Rad53 signaling named DAMP (Dampens Adaptor MediatedPhosphosignaling). DAMP relies on the Slx4 scaffold protein competing with the checkpoint adaptor Rad9 at sites of lesions to counteract Rad53 activation. Here, I show that DAMP functions in parallel with canonical phosphatase mechanisms for Rad53 down-regulation. I show that slx4 cells phenocopy cells lacking the main Rad53 phosphatase, Pph3. Both pph3[DELTA] and slx4[DELTA] cells show selective sensitivity to methyl methanesulfonate (MMS), accumulate chromosomal defects and hyperactivate Rad53. Both Slx4 and Pph3 seem to converge to the regulation of the Mus81 nuclease, which is necessary for downstream repair. Interestingly, deletion of both SLX4 and PPH3 leads to a synergistic increase in MMS sensitivity and Rad53 activation, suggesting that efficient down-regulation of DDC signaling requires the coordinated action of DAMP and phosphatases. I propose that these mechanisms operate in distinct spatio-temporal modes, with Slx4 dampening Rad53 activation at lesions, and Pph3 functioning on free, active pools of Rad53 to turn off the checkpoint response. Collectively, the results from my thesis reveal that checkpoint control is tightly regulated by multiple coordinated mechanisms to allow for cell proliferation following DNA damage. Furthermore, given that checkpoint regulation is highly conserved from yeast to humans, this work contributes to better understanding the importance of checkpoint control and provides rationale to manipulate checkpoint signaling in human systems and particularly in cancer research