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Rad53 is an essential protein kinase governing DNA damage and replication stress checkpoints in budding yeast. It also appears to be involved in cellular morphogenesis processes. Mass spectrometry analyses revealed that Rad53 is phosphorylated at multiple SQ/TQ and at SP/TP residues, which are typical consensus sites for phosphatidylinositol 3-kinase-related kinases and CDKs, respectively. Here we show that Clb-CDK1 phosphorylates Rad53 at Ser(774) in metaphase. This phosphorylation event does not influence the DNA damage and replication checkpoint roles of Rad53, and it is independent of the spindle assembly checkpoint network. Moreover, the Ser-to-Asp mutation, mimicking a constitutive phosphorylation state at site 774, causes sensitivity to calcofluor, supporting a functional linkage between Rad53 and cellular morphogenesis.

The use of translesion synthesis (TLS) polymerases to bypass DNA lesions during replication constitutes an important mechanism to restart blocked/stalled DNA replication forks. Because TLS polymerases generally have low fidelity on undamaged DNA, the cell must regulate the interaction of TLS polymerases with damaged versus undamaged DNA to maintain genome integrity. The Saccharomyces cerevisiae checkpoint proteins Ddc1, Rad17, and Mec3 form a clamp-like structure (the 9-1-1 clamp) that has physical similarity to the homotrimeric sliding clamp proliferating cell nuclear antigen, which interacts with and promotes the processivity of the replicative DNA polymerases. In this work, we demonstrate both an in vivo and in vitro physical interaction between the Mec3 and Ddc1 subunits of the 9-1-1 clamp and the Rev7 subunit of the Polzeta TLS polymerase. In addition, we demonstrate that loss of Mec3, Ddc1, or Rad17 results in a decrease in Polzeta-dependent spontaneous mutagenesis. These results suggest that, in addition to its checkpoint signaling role, the 9-1-1 clamp may physically regulate Polzeta-dependent mutagenesis by controlling the access of Polzeta to damaged DNA.

The cellular response to DNA lesions entails the recruitment of several checkpoint and repair factors to damaged DNA, and chromatin modifications may play a role in this process. Here we show that in Saccharomyces cerevisiae epigenetic modification of histones is required for checkpoint activity in response to a variety of genotoxic stresses. We demonstrate that ubiquitination of histone H2B on lysine 123 by the Rad6-Bre1 complex, is necessary for activation of Rad53 kinase and cell cycle arrest. We found a similar requirement for Dot1-dependent methylation of histone H3. Loss of H3-Lys(79) methylation does not affect Mec1 activation, whereas it renders cells checkpoint-defective by preventing phosphorylation of Rad9. Such results suggest that histone modifications may have a role in checkpoint function by modulating the interactions of Rad9 with chromatin and active Mec1 kinase.

Rad17-Mec3-Ddc1 forms a proliferating cell nuclear antigen-like complex that is required for the DNA damage response in Saccharomyces cerevisiae and acts at an early step of the signal transduction cascade activated by DNA lesions. We used the mec3-dn allele, which causes a dominant negative checkpoint defect in G1 but not in G2, to test the stability of the complex in vivo and to correlate its assembly and disassembly with the mechanisms controlling checkpoint activation. Under physiological conditions, the mutant complex is formed both in G1 and G2, although the mutant phenotype is detectable only in G1, suggesting that is not the presence of the mutant complex per se to cause a checkpoint defect. Our data indicate that the Rad17-Mec3-Ddc1 complex is very stable, and it takes several hours to replace Mec3 with Mec3-dn within a wild type complex. On the other hand, the mutant complex is rapidly assembled when starting from a condition where the complex is not pre-assembled, indicating that the critical factor for the substitution is the disassembly step rather than complex formation. Moreover, the kinetics of mutant complex assembly, starting from conditions in which the wild type form is present, parallels the kinetics of checkpoint inactivation, suggesting that the complex acts in a stoichiometric way, rather than catalytically.