An in vitro DNA replication system using yeast 2-Mum and ARS (autonomously replicating sequences) plasmid DNAs, developed in this laboratory, has been used to investigate the mechanism of DNA replication in yeast. To identify and purify enzymes and components required for yeast chromosomal DNA replication, the crude extract system has been fractionated and reconstituted with the help of several temperature-sensitive chromosomal DNA replication mutants. To aid in overproducing and purifying such DNA replication proteins, the DBF1 and 2 genes (which are required for the elongation step of DNA replication) and the TS26 gene (required for the initiation of DNA replication) have been cloned, their nucleotide sequences determined, antibodies to them raised and their regulation studied. lt was found that the DBF2 protein is a serine/threonine-specific protein kinase, suggesting that protein phosphorylation regulates elongation of DNA synthesis in yeast. Finally, yeast DNA polymerases I, II, and III (The last being newly discovered in yeast crude extracts) have been purified to homogeneity for the first time and studied extensively. Using inhibitors and antibodies against each purified DNA polymerase, it has been established that each polymerase is unique and has a different function in yeast cells. In order to study these functions, the molecular cloning of DNA polymerases I, II and III is underway using either antibodies or the amino acid sequences Df oligopeptides generated from the purified polymerases. In the meantime, mammalian PCNA/cycling, which is under cell-cycle control and is a subunit of DNA polymerase delta, has been shown to stimulate the yeast DNA polymerase III reaction, suggesting that yeast DNA polymerase III is a homolog of mammalian DNA polymerase delta.
