Studies on a cell division cycle (cdc) mutant of the yeast Saccharomyces cerevisiae, defective in the initiation of DNA synthesis
These studies were undertaken in an attempt to determine the nature of the defect in a temperature-sensitive yeast cell division cycle (cdc) mutant, cdc7.4, which defines an essential step immediately prior to DNA synthesis. The in vivo characterisation of this mutant was extended. A yeast mating pheromone, α-factor, was purified and used for cell -synchronization purposes. Functional CDC 7 gene product was found to be necessary for nuclear and 2μn plasmid DNA replication, but not for mitochondrial DITA replication, which was unaffected by α-factor. RNA, protein, phospholipid and phosphoprotein syntheses were shown to be unaffected by the mutation. The in vivo characterisation work suggested that the cdc7.4 mutation was due to a temperature-sensitive protein. One- and two-dimensional polyacrylamide gel electrophoresis were used to detect mutationally altered proteins. An abnormal protein present in the original cdc7.4 strain was shown to be unrelated to the lesion in DNA synthesis by a genetic analysis. A second temperature-sensitive defect was also discovered in this strain. An extensively characterised nuclear fraction was analysed on SDS-polyacrylamide gels, and another abnormal band pattern was observed. Various attempts were made to construct in vitro DNA synthesising systems. Controlled lysis of spheroplasts and the fusion of protein-filled liposomes with cdc7.4 spheroplasts were both nominally successful, but were considered unpractical. Reconstitution of a system from purified components was undertaken. A hybrid plasmid (pJDB219) containing yeast 2μn DNA sequences was used as the substrate. Several proteins implicated in DNA replication in other systems were partially purified, including DNA, polymerases, RNA polymerases, single-stranded DNA binding protein and DNA topoisomerase. These proteins, and partially fractionated endonuclease activity, were shown not to be thermosensitive.