The control of proliferation of animal cells during normal development and the loss of control in the conversion of normal cells to cancer cells is of central importance in human growth, aging and disease. In order for cells to divide they must first replicate their DNA, and the control of initiation of DNA replication is closely coupled to the control of cell division in eukaryotic systems. Understanding the molecular mechanism of DNA replication in eukaryotic cells is fundamental to gaining an understanding of these control systems. To gain a better understanding of the process of DNA replication in eukaryotic cells, I propose to continue a study of the replication apparatus of the yeast Saccharomyces cerevisiae. The objects of the study are small circular DNA plasmids which replicate in yeast under nuclear control. Both endogenous yeast plasmids and recombinant DNA plasmids containing functional yeast replicons cloned in Escherichia coli vectors will be used. Assays will be developed for the proteins involved in the replication process, the proteins will be purified using established biochemical techniques, and the mechanisms of action of the purified proteins will be investigated. Mutant plasmids will be constructed and the effects of the mutations on the replication of the plasmids in yeast and in test tube will be evaluated in comparison with the unmodified templates. I will also investigate the influence of yeast mutations involved with host cell DNA synthesis on the duplication of the plasmids containing yeast chromosomal replicons. Replication extracts from the host mutant strains will be prepared and correlation of the mutations with specific replication proteins will be attempted. The influence of drugs and hormones known to affect DNA replication will be measured using the cell-free replication system.