Accumulation of Replicative DNA Intermediates in Tetrahymena after Excision‐Repairable Damage to DNA

Abstract

Inhibition of DNA synthesis in the eukaryotic organism Tetrahymena pyriformis by thymine starvation results in the formation and accumulation of a distinct class of DNA during the first hour after re-initiation of the synthesis. This DNA, when synthesized in the presence of 5-bromodeoxyuridine (BrdUrd), bands as a distinct peak at an intermediate density of (see article) in a neutral CsCl buoyant density gradient. Following short pulses of BrdUrd, the DNA of intermediate density accounts for up to 90% of the newly synthesized DNA and binding assays on nitrocellulose filters show the presence of single-stranded regions. Alkaline buoyant density and alkaline velocity gradients clearly demonstrate the presence of non-covalently linked newly synthesized fragments with an average length shorter than that of the parental strands. Pulse experiments show that the DNA of intermediate density is progressively converted to fully hybrid (light - heavy) DNA molecules. It is therefore suggested that in response to damage caused by thymine starvation, a replicative DNA intermediate accumulates at the growing point. A similar phenomenon has also been observed after irridation with ultraviolet light. The effect of the protein synthesis inhibitor cycloheximide on the formation on the intermediate and the fully hybrid material is also described.