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

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In order to establish an in vivo function of Saccharmoyces cerevisiae DNA polymerase II (a probable homologue of mammalian DNA polymerase epsilon) various temperature-sensitive II mutants were isolated by the plasmid shuffling method. It has been shown that the temperature-sensitive mutants (ts9, 18, and 31) have not only temperature-sensitive DNA polymerase II activity, but also exhibit temperature-sensitive chromosomal replication in vivo. Furthermore, 3'-5' exonuclease-deficient DNA polymerase II mutants exhibit high spontaneous mutation frequency, although chromosomal replication is not affected by these mutations. These results are consistent with the notion that DNA polymerase II participates in chromosomal replication in yeast. These results strongly support our current three DNA polymerase model that is able to explain how the three DNA polymerases participate in yeast chromosomal replication. The CDC7 gene is required for the last step before initiation of chromosomal replication in the yeast Saccharomyces cerevisiae. To understand the regulatory mechanism of initiation of chromosomal replication, we have isolated the gene on a multicopy plasmid which suppresses temperature-sensitive cdc7-1. This gene is identical to DBF4 which we have previously isolated by complementation with a cell cycle mutant dbf4. Furthermore, we find that CDC7 on a multicopy plasmid also suppresses temperature-sensitive dbf4-l and dbf4-2. From these results, we concluded that DBF4 gene product directly interacts with CDC7 gene product (a serine-threonine protein kinase). Finally, we have isolated and sequenced three new genes which suppress temperature-sensitive dbf4-1 mutant cells and characterized them extensively.