| The DNA replication checkpoint, which is present in all eukaryotes, supervises DNA synthesis during S phase and protects genome integrity when DNA replication is perturbed. Activation of the replication checkpoint requires Mrc1, the mediator of replication checkpoint, to channel the signal from the sensor kinase Rad3 to the downstream effector kinase Cds1. Once activated, the replication checkpoint stimulates cellular responses, such as stabilization of arrested replication forks and increased dNTP production rate to counteract the perturbations.;Chapter I presents the discovery of mrc1+ and SPAP14E8.02, two novel Cdc10-regulated genes specifically expressed during S phase of the cell cycle in S. pombe, by screening 25 candidate genes uncovered by a bioinformatic analysis of the genome. Although Mrc1 and SPAP14E8.02 are not essential during the normal cell cycle, Mrc1 was found to be required for replication checkpoint activation when DNA replication is arrested.;Chapter II describes analysis by genetic and biochemical approaches of the molecular mechanism of Mrcl in the activation of the effector kinase Cds1. I found that the sensor kinase Rad3 phosphorylates two TQ motifs in short repeated sequences of Mrc1. The phosphorylated TQ motifs function redundantly in recruiting Cds1 by binding its FHA domain. Once bound to Mrc1, Cds1 is phosphorylated by Rad3, which primes Cds1 for autoactivation. The primed Cds1 molecules dimerize via phospho-specific interactions mediated by the FHA domains and are activated by autophosphorylation in trans of the kinase domains.;Chapter III focuses on the biochemical mechanism of Cds1 autoactivation. In part IIIA, I describe the identification of three potential phosphorylation sites, T328, T332, and Y352, among all serine, threonine and tyrosine residues in the kinase domain of Cds1 that are critical for Cds1 activation. Among the three potential phosphorylation sites, T328 is autophosphorylated in vitro as determined by phosphopeptide mapping and phospho-specific antibodies. Phosphorylation of T328 strongly correlates with the activation of Cds1 in vitro. T328 resides in the "activation segment" of the kinase domain and its phosphorylation is probably essential for kinase activation.;Large scale purification of Cds1 preparatory to structural studies is presented in part IIIB of Chapter III. Cds1 was successfully purified to apparent homogeneity from E. coli in milligram quantities by co-expression with λ-phosphatase. Cds1 mutant proteins including the isolated kinase domain were also purified to homogeneity. |
