The Saccharomyces cerevisiae CDC7 protein kinase, a potential link between START and the initiation of DNA replication

Citation

Yoon, Hye-Joo (1992) The Saccharomyces cerevisiae CDC7 protein kinase, a potential link between START and the initiation of DNA replication. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/mapz-nx18. https://resolver.caltech.edu/CaltechTHESIS:09152011-110250530

Abstract

Two major controls of the eukaryotic cell cycle exist in the G1/S and G2/M transitions. In an attempt to understand the molecular basis of the G1/S regulatory pathway, biochemical characterization of the Saccharomyces cerevisiae CDC7 protein has been carried out. The temperature sensitive cdc7 mutation arrests yeast cells at the G1/S phase boundary, after the completion of START and prior to the initiation of DNA synthesis. START is defined as the point of commitment to DNA replication in yeast. The open reading frame encoding CDC7 protein contains the 11 catalytic domains characteristic of protein kinases. That CDC7 protein indeed has protein kinase activity was demonstrated by incubating CDC7 immune complexes with histone H1. Kinase activity was elevated more than 10-fold in strains carrying the wild-type CDC7-overproducing plasmid. Overproduction of thermolabile cdc7 protein gave rise to overproduction of thermolabile kinase. In vivo _(32)P-labeling of yeast cells revealed that CDC7 protein itself was a phosphoprotein. In addition, indirect immunofluorescence and biochemical fractionation studies showed CDC7 was located mainly in yeast nuclei.

The cell cycle-specific roles of CDC7 at the G1/S border were explored by following the phosphorylation state and kinase activity of CDC7 protein throughout the cell cycle. CDC7 was not active as a histone H1 kinase, nor was it phosphorylated in cells arrested by a cdc28 mutation. Furthermore, phosphorylation of CDC7 protein was required for its activity. CDC28 encodes the budding yeast homolog of the highly conserved p34^(cdc2)/MPF kinase subunit and its function is crucial to passage through START. The possibility that CDC7 is a substrate of the CDC28 kinase was tested using CDC28 immune complexes and bacterially produced, inactive CDC7 kinase. The CDC28 kinase specifically phosphorylated the CDC7 protein. which in tum led to activation of the CDC7 kinase. CDC28 and CDC7 could interact in vivo as well since CDC7 hyperexpression suppressed the inviability of a cdc28 mutation at 33°C but not at 36°C. Finally, possible mechanisms of action of CDC7 protein kinase in the initiation of DNA synthesis were examined by searching for endogenous substrates among yeast replication proteins. The 34 kDa subunit of yeast replication protein-A (RP-A) is a substrate of both CDC7 and CDC28 kinases.

In conclusion, the biochemical evidence indicates that there is a phosphorylation cascade operating between START and the initiation of DNA replication involving yeast CDC28, CDC7, and RP-A.

Thesis Files