The C-terminal residues of Saccharomyces cerevisiae Mec1 are required for its localization, stability, and function.

Mec1, a member of the phosphoinositide three-kinase-related kinase (PIKK) family of proteins, is involved in the response to replicative stress and DNA damage and in telomere maintenance. An essential 30 to 35 residue, the FATC domain is found at the C-terminus of all PIKK family members. To investigate the roles of the C-terminal residues of Mec1, we characterized alleles of Saccharomyces cerevisiae mec1 that alter the FATC domain. A change of the terminal tryptophan to alanine resulted in temperature-sensitive growth, sensitivity to hydroxyurea, and diminished kinase activity in vitro. Addition of a terminal glycine or deletion of one, two, or three residues resulted in loss of cell viability and kinase function. Each of these Mec1 derivatives was less stable than wild-type Mec1, eluted abnormally from a size exclusion column, and showed reduced nuclear localization. We identified rpn3-L140P, which encodes a component of the 19S proteasomal regulatory particle of the 26S proteasome, as a suppressor of the temperature-sensitive growth caused by mec1-W2368A. The rpn3-L140P allele acted in a partially dominant fashion. It was not able to suppress the inviability of the C-terminal truncations or additions or the hydroxyurea sensitivity of mec1-W2368A. The rpn3-L140P allele restored Mec1-W2368A to nearly wild-type protein levels at 37°, an effect partially mimicked by the proteasome inhibitor MG-132. Our study supports a role for the C-terminus in Mec1 folding and stability, and suggests a role for the proteasome in regulating Mec1 levels.

A quantitative model of the initiation of DNA replication in Saccharomyces cerevisiae predicts the effects of system perturbations.

BACKGROUND: Eukaryotic cell proliferation involves DNA replication, a tightly regulated process mediated by a multitude of protein factors. In budding yeast, the initiation of replication is facilitated by the heterohexameric origin recognition complex (ORC). ORC binds to specific origins of replication and then serves as a scaffold for the recruitment of other factors such as Cdt1, Cdc6, the Mcm2-7 complex, Cdc45 and the Dbf4-Cdc7 kinase complex. While many of the mechanisms controlling these associations are well documented, mathematical models are needed to explore the network's dynamic behaviour. We have developed an ordinary differential equation-based model of the protein-protein interaction network describing replication initiation. RESULTS: The model was validated against quantified levels of protein factors over a range of cell cycle timepoints. Using chromatin extracts from synchronized Saccharomyces cerevisiae cell cultures, we were able to monitor the in vivo fluctuations of several of the aforementioned proteins, with additional data obtained from the literature. The model behaviour conforms to perturbation trials previously reported in the literature, and accurately predicts the results of our own knockdown experiments. Furthermore, we successfully incorporated our replication initiation model into an established model of the entire yeast cell cycle, thus providing a comprehensive description of these processes. CONCLUSIONS: This study establishes a robust model of the processes driving DNA replication initiation. The model was validated against observed cell concentrations of the driving factors, and characterizes the interactions between factors implicated in eukaryotic DNA replication. Finally, this model can serve as a guide in efforts to generate a comprehensive model of the mammalian cell cycle in order to explore cancer-related phenotypes.