Genetic analysis of the in vivo role of DNA polymerases in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, the suggested functions of DNA polymerases (DNApol) have been based primarily on the characterization of the wild-type and mutant enzymes via in vitro studies. Here we describe a novel replication system to decipher the role of different DNA polymerases in in vivo DNA replication. Using this system, [α-³²P]dNTP is allowed to cross the membrane of permeabilized cells; then the nature of the radiolabeled products of DNA synthesis is analyzed by gel electrophoresis and densitometry. Results of such analyses show that these replication intermediates are synthesized in the range 50–1,300 bp, which are then rapidly elongated and then ligated into longer DNA chains, and that the in vivo synthesis of yeast DNA fragments is dependent essentially on DNApolα and DNApolδ, but not necessarily on DNApolɛ. Results presented here support the views that DNApolɛ is dispensable for yeast DNA replication or that DNA polα and DNApolδ are epistatic to DNApolɛ in yeast.