Deoxyribonucleic Acid Synthesis in Permeabilized Spheroplasts of Saccharomyces cerevisiae

Abstract

Osmotically shocked spheroplasts from S. cerevisiae incorporated deoxynucleoside triphosphates specifically into double-stranded nuclear and mitochondrial DNA. Results with this in vitro system for cells with and without mitochondrial DNA were compared. Strains lacking mitochondrial DNA were used to study nuclear DNA replication. With a temperature-sensitive mutant defective in DNA replication in vivo, DNA synthesis in vitro was temperature-sensitive as well. The product of synthesis with all strains after very short labeling times consisted principally of short fragments that sedimented at approximately 4S in alkali; with longer pulse times or a chase with unlabeled nucleotides, they grew to a more heterogenous size, with an average of 6-8S and a maximum of 15S. There was little, if any, integration of these DNA fragments into the high MW nuclear DNA. Analysis of CsCl density gradient centrifugation after incorporation of bromodeoxyuridine triphosphate showed that most of the product consisted of chains containing both preexisting and newly synthesized material, but there was also a small fraction (about 20%) in which the strands were fully synthesized in vitro. 32P-label transfer (nearest-neighbor) experiments demonstrated that at least a part of the material synthesized in vitro contained RNA-DNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking up TMP, sedimented in alkali at 4S, as in the case of the in vitro experiments.