The enzymology of DNA replication has shown a marked increase in complexity. As this multitude of proteins expands, it becomes clear that many perform as accessory factors which contribute to the """"""""correct"""""""" or """"""""faithful"""""""" functioning of the primary enzymes. The plan outlined here proposes to study DNA replication in yeast with particular emphasis on the faithful duplication of the gene. The purpose of this research proposal is the identification, purification and characterization of yeast DNA replication proteins. An in vitro replication system will be established to study DNA synthesis mutants in crude extracts and to assay replication proteins by complementation of mutant extracts. The cloning of replication genes will aid the identification and purification of the proteins coded for by these genes. In addition, antibodies can be prepared against segments of proteins specified by cloned genes. The genes for the proteins which bind the DNA synthesis inhibitor, aphidicolin will also be cloned and antibodies prepared. These techniques will be used in conjunction with conventional assays to prepare yeast DNA replication enzymes. Reconstitution of the enzyme complexes responsible for the orderly duplication of the genome can then be attempted.
