HAIRPIN STRUCTURES ARE THE PRIMARY AMPLIFICATION PRODUCTS - A NOVEL MECHANISM FOR GENERATION OF INVERTED REPEATS DURING GENE AMPLIFICATION

Early events of DNA amplification which occur during perturbed replica tion were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contai n molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cell s. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several ex perimental procedures such as sucrose gradients, ''snap-back'' assay, and two-dimensional gel electrophoresis, we show that the overreplicat ed DNA contains IRs which are synthesized de novo as hairpins or stem- loop structures which were detached from the template molecules. The f ully replicated SV40 molecules synthesized bg the HeLa extracts do not contain such IRs. We propose ''U-turn replication'' as a novel mechan ism for gene amplification, accounting for the generation of extrachro mosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.