Carcinogen-Induced DNA Amplification In Vitro: Overreplication of the Simian Virus 40 Origin Region in Extracts from Carcinogen-Treated CO60 Cells

Abstract

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.

Hairpin Structures Are the Primary Amplification Products: a Novel Mechanism for Generation of Inverted Repeats during Gene Amplification

“…Cellular factors induced by the carcinogen treatment were shown to be responsible for the amplification process (5). To identify these factors and to elucidate the structure of the initial amplification products, we have constructed an in vitro system for SV40 DNA amplification based on the in vitro SV40 replication system developed by Li and Kelly (31).…”