Co-localization of Sensors is Sufficient to Activate the DNA Damage Checkpoint in the Absence of Damage

Previous work on the DNA damage checkpoint in Saccharomyces cerevisiae has shown that two complexes independently sense DNA lesions: the kinase Mec1-Ddc2 and the PCNA-like 9-1-1 complex. To test whether co-localization of these components is sufficient for checkpoint activation, we fused these checkpoint proteins to the LacI repressor and artificially co-localized these fusions by expressing them in cells harboring Lac operator arrays. We observed Rad53 and Rad9 phosphorylation, Sml1 degradation and metaphase delay, demonstrating that co-localization of these sensors is sufficient to activate the checkpoint in the absence of DNA damage. Our tethering system allowed us to examine checkpoint activation in the absence of damage processing and checkpoint protein recruitment. Thus, we were able to establish that CDK functions in the checkpoint pathway downstream of damage recognition. We found that CDK-dependent phosphorylation of Rad9 is required for efficient checkpoint activation. Lastly, we began analysis of the regulation by CDK on Rad9 in its ability to activate Rad53. We found that the Rad9 CDK mutant was not phosphorylated by Mec; and was not able to find Rad53 efficiently after DNA damage