MECHANISM OF DNA REPLICATION IN EUCARYOTES--YEAST AS A MODEL SYSTEM

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Various temperature-sensitive DNA polymerase II mutants of S. cerevisiae have been characterized. The purified DNA polymerase II from the mutants exhibited temperature-sensitive DNA polymerase activity, while in vivo chromosomal DNA synthesis was greatly reduced at the restrictive temperatures. Furthermore, these mutant cells had a single-base-pair change which generates a single amino acid change in the DNA polymerase II gene. These results proved that S. cerevisiae DNA polymerase II participates in its chromosomal DNA replication. By using the PCR technique, we have cloned the DNA polymerase gene which has a high homology to S. cerevisiae DNA polymerase II from a fission yeast S. pombe and from Drosophila melanogaster and their partial nucleotide sequences have been determined. The CDC7 gene product is required for the last step before initiation of chromosomal DNA replication in S. cerevisiae. In the previous year, we have shown genetically that the CDC7 gene product (a protein kinase) directly interacts with the DBF4 gene product which is also required for initiation of chromosomal DNA replication and suggested that the DBF4 protein activates the protein(s) which is needed for initiation of chromosomal DNA replication. In this year, we have obtained biochemical evidences for the direct interaction between the DBF4 protein and the CDC7 protein. Furthermore, an active form of the CDC7 protein kinase consisted of the CDC7 and DBF4 polypeptides. These data further substantiated our proposal that the DBF4 protein works as a CDC28 (CDC2) cyclin and modulates activity of the CDC7 protein kinase. Finally, we have isolated, sequenced, and characterized the CDC5 gene which interacts with DBF4, CDC15, and DBF2 gene products.