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In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed(More)
CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven(More)
Eukaryotic cells ensure the stable propagation of their genome by coupling each round of DNA replication (S phase) to passage through mitosis (M phase). This control is exerted at the initiation of replication, which occurs at multiple origins throughout the genome. Once an origin has initiated, reinitiation is blocked until the completion of mitosis,(More)
The initiation of DNA replication in Saccharomyces cerevisiae requires the protein product of the CDC45 gene. We report that although Cdc45p is present at essentially constant levels throughout the cell cycle, it completes its initiation function in late G1, after START and prior to DNA synthesis. Shortly after mitosis, cells prepare for initiation by(More)
As phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) on Ser51 inhibits protein synthesis, cells restrict this phosphorylation to the antiviral protein kinase PKR and related eIF2α kinases. In the crystal structure of the PKR-eIF2α complex, the C-terminal lobe of the kinase contacts eIF2α on a face remote from Ser51, leaving Ser51 ∼ 20 Å(More)
Invasive human breast carcinomas frequently coexpress increased hepatocyte growth factor (HGF) and its receptor Met, suggesting that establishment of an autocrine HGF loop is important in malignant disease. This study examines the expression patterns of HGF and Met activation during tumorigenesis and metastasis using a MCF10A-based model of Ha-Ras-induced(More)
RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and, as a result, undesirably stimulate RAS/ERK(More)
The ability of protein kinases to switch between inactive and active states is critical to control the outputs of cellular signaling pathways. In several protein kinases, the conformation of helix αC is a key hub on which regulatory inputs converge to induce catalytic switching. An emerging mechanism involved in regulating helix αC orientation is the(More)
Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase(More)
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