Amplification of a plasmid bearing a mammalian replication initiation region in chromosomal and extrachromosomal contexts

Amplified genes in cancer cells reside on extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSRs). We used a plasmid bearing a mammalian replication initiation region to model gene amplification. Recombination junctions in the amplified region were comprehensively identified and sequenced. The junctions consisted of truncated direct repeats (type 1) or inverted repeats (type 2) with or without spacing. All of these junctions were frequently detected in HSRs, whereas there were few type 1 or a unique type 2 flanked by a short inverted repeat in DMs. The junction sequences suggested a model in which the inverted repeats were generated by sister chromatid fusion. We were consistently able to detect anaphase chromatin bridges connected by the plasmid repeat, which were severed in the middle during mitosis. De novo HSR generation was observed in live cells, and each HSR was lengthened more rapidly than expected from the classical breakage/fusion/bridge model. Importantly, we found massive DNA synthesis at the broken anaphase bridge during the G1 to S phase, which could explain the rapid lengthening of the HSR. This mechanism may not operate in acentric DMs, where most of the junctions are eliminated and only those junctions produced through stable intermediates remain.