An in vitro DNA replication system of yeast 2-Mum and ARS1 plasmid DNAs has been used as a model to investigate the mechanism of DNA replication in eucaryotes. To identify various enzymes and protein factors required for DNA replication, the system has been further fractionated and reconstituted by using separated fractions. From these experiments, two additional proteins have been identified and purified. These are newly identified single-stranded DNA-dependent ATPase (ATPase III) and DNA topoisomerase II and have been extensively studied in this year. The ATPase III has additional enzymatic activity which unwinds double-stranded DNA utilizing energy of ATP hydrolysis (helicase activity). Furthermore, RNase Hs and single-stranded DNA binding proteins which might participate in DNA replication have been purified to homogeneity by following their enzymatic activities. To provide that these proteins are required for yeast DNA replication, antibodies have been raised aginst each and their genes have been identified and cloned from lambda gt11 expression yeast genomic library using the antibodies. Their nucleotide sequences have been determined and the genes have been mapped on the chromosomes. Finally, the essentiality of each gene has been tested by gene disruption and one of single-stranded DNA binding proteins (20k dalton) is found to be required for yeast cell viability. In order to permit identification and isolation of other DNA replication proteins, new temperature-sensitive DNA replication mutants of yeast have been isolated, characterized genetically, some of these mutant genes have been cloned and their nucleotide sequences have been determined. Currently, to ease the purification of their gene products overproduction of the gene products is being carried out.
