Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (C060) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated C060 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.Genetic instability is a hallmark of tumor cells but occurs rarely in normal mammalian cells (13,39,40,51,53,60). One of the predominant manifestations of genetic instability is gene amplification (for a review, see reference 48). Many studies have shown that gene amplification is a dynamic process and that the amplified sequences can be associated with the chromosome, appearing as expanded chromosomal regions, or they can be extrachromosomal, thus represented as acentric circular structures known as double minute chromosomes (for reviews, see references 6 and 58). The amplification units are often organized as inverted tandem repeats (3, 14, 15, 18, 20, 25, 34-36, 38, 41, 44-46, 49). It has been suggested that the formation of inverted duplications is involved in the generation of amplified DNA (14,15,20,41,44,45) and thus might be the initial step in the amplification process.The diversity in size and molecular configurations suggests the existence of a number of distinct mechanisms by which gene amplification occurs (for reviews, see references 36 and 58). For example, it could result from overreplication of specific sequences, from unequal segregation of recombination and replication products to the daughter cells, or from a combination of both. In most systems used to study gene amplification, the amplified products were investigated several generations after the initial amplification event, and therefore analysis of the primary amplification products and understanding of the mode of DNA amplification were not possible. Hence, the question of amplification is still open. Amplification can be enhanced by carcinogens and drugs arresting DNA replication (7,12,16,17,37,42,52). Furthermore, changes in the cell cycle, such as uncontrolled entry into S phase or arrest of cells in S phase, may lead to aberrant DNA replication, which was suggested to be involved in the process o...