The CDC7 protein of Saccharomyces cerevisiae is a phosphoprotein that contains protein kinase activity.

Abstract

The CDC7 protein of Saccharomyces cerevisiae may be involved in the G1/S-phase transition and/or in the initiation of mitotic DNA synthesis. The CDC7 gene has two in-frame AUG codons as possible translation start sites, which would produce 58-and 56-kDa proteins, respectively. Both p58 and p56 derived from recombinant plasmids complement the temperature-sensitive growth defect of the cdc7-1 allele. To determine the biochemical function of the CDC7 protein, the CDC7 gene was cloned and polyclonal antibodies were produced against the CDC7 protein. CDC7 immune complexes prepared from yeast with these antibodies phosphorylate histone H1. Kinase activity is thermolabile in strains carrying the cdc7-1 temperature-sensitive mutant allele and is elevated >10-fold in strains carrying plasmids overexpressing either p56 or p58, confirming that the kinase in the immunoprecipitates is the CDC7 gene product. In addition, we show that CDC7 is a phosphoprotein itself. Indirect immunofluorescence and biochemical fractionation show that the CDC7 protein is present at relatively high concentrations in the nucleus compared with the cytoplasm, suggesting that nuclear proteins may be substrates for the CDC7 protein.Two major events define the eukaryotic cell cycle: replication of the chromosomes during S phase and segregation of the chromosomes during mitosis. Replication and segregation are separated by two gap periods, G1 and G2, during which the cells prepare for these events. There are also two major control points for the cell cycle in G1 and G2, the point of commitment to DNA synthesis in G1 and the regulation of progression into mitosis in G2. Although we have made great strides in understanding mitotic control in the past three years, relatively little is known about the events in G1 that lead to S phase. In yeast, the point of commitment to DNA synthesis is defined as "Start", early in G1. DNA synthesis does not ensue immediately after Start, however, and it is not clear whether the lag is due to assembly of the replication apparatus or to a cascade of controls initiated at Start. CDC28, CDC4, and CDC7 define a dependent series of events set in motion at Start (1-3). CDC28 encodes a protein kinase subunit, the analog of the highly conserved cdc2+/ MPF kinase subunit involved in regulation of mitosis (3).CDC4 is homologous to one subunit of the signal transducing protein, transducin (4). The DNA sequence of the CDC7 gene predicts a protein that has homology to catalytic domains of the protein kinases CDC2/CDC28, NIM1, and a number of kinase-related transforming proteins (5). The CDC7 sequence differs from that of other protein kinases in the so-called phosphate acceptor domain, however (5, 6). We have been interested in the role of CDC7 in the events linking Start and the initiation of DNA replication. Mutations in CDC7 appear to block a precondition for DNA synthesis because cells carrying these lesions cannot start new rounds of DNA replication after a shift from permissive to nonpermissive temperature (7,8). The cd...

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