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

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In order to establish an in vivo function of Saccharomyces cerevisiae DNA polymerase II (a homolog of mammalian DNA polymerase epsilon) which we have identified and purified last year, the gene has been identified, cloned by both antiserum against the purified DNA polymerase II and its partial amino acid sequence and its nucleotide sequence have been determined. Mutants having various deletions and disrupted gene have been generated. Using these mutants, it has been shown that DNA polymerase II is an essential enzyme for yeast chromosome replication and it is conceivable that DNA polymerase epsilon could be an essential enzyme for DNA replication in mammalian cells. Based upon these results, we have proposed a new model to explain how eucaryotic chromosome is replicated by three DNA polymerases inside the cells. We also have cloned and sequences the genes encoding DNA polymerase II accessory proteins (subunits B, C, and D). CDC7 gene is well known to be required for initiation of chromosome replication, but not an origin-specific binding protein. We have isolated and sequenced one gene which is able to suppress cdc7 mutations. This gene is an already known cell-division-cycle gene, DBF4, which we have been studying independently from the above mentioned. From this, we have concluded that the DBF4 protein interacts directly with the CDC7 protein (associated with a protein kinase activity) to facilitate initiation of chromosome replication during S-phase in S. cerevisiae. Another DNA replication protein that we have studied in this year is a new type of single-stranded DNA binding protein which consists of three different polypeptides. This protein complex is a homolog of human RF-A protein. This protein greatly stimulates DNA polymerase I reaction, but has little effect on either DNA polymerase II or III reaction. Most of the protein localizes in yeast nuclear matrix, suggesting that it plays a very important role for chromosome replication.