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.

We previously found that a plasmid bearing a replication initiation region efficiently initiates gene amplification in mammalian cells and that it generates extrachromosomal double minutes and/or chromosomal homogeneously staining regions. During analysis of the underlying mechanism, we serendipitously found that hairpin-capped linear DNA was stably maintained as numerous extrachromosomal tiny episomes for more than a few months in a human cancer cell line. Generation of such episomes depended on the presence of the replication initiation region in the original plasmid. Despite extrachromosomal maintenance, episomal gene expression was epigenetically suppressed. The Southern blot analysis of the DNA of cloned cells revealed that the region around the hairpin end was diversified between the clones. Furthermore, the bisulfite-modified PCR and the sequencing analyses revealed that the palindrome sequence that derived from the original hairpin end or its end-resected structure were well preserved during clonal long term growth. From these data, we propose a model that explains the formation and maintenance of these episomes, in which replication of the hairpin-capped DNA and cruciform formation and its resolution play central roles. Our findings may be relevant for the dissection of mammalian replicator sequences.Gene amplification plays a pivotal role in mammalian malignant transformation through acquisition of growth advantages or drug resistances (for recent reviews, see Refs. 1-4). Amplified genes are cytogenetically detected at extrachromosomal double minutes (DMs) 2 or in chromosomal homogeneously staining region (HSRs). DMs are acentric, atelomeric chromatin bodies composed of amplified DNA of genomic origin. We previously found that a plasmid with a mammalian replication initiation region (IR) and a matrix attachment region (MAR) is efficiently amplified to high copy number in mammalian cells and that it generates DMs and/or HSRs composed of plasmid sequences (5, 6). Because IRs and MARs are scattered throughout the mammalian genome in a frequency of once per several tens of kilobase pairs and because it was proposed that circular molecules excised from the genome may mediate gene amplification in cancer cells (7, 8), we considered the plasmid system as a novel one that efficiently reproduces gene amplification in cultured cells. Thus, we examined the mechanism by which the IR/MAR plasmid mimics gene amplification (5, 9). We suggested that the IR/MAR plasmid undergoes multiplication to produce a large extrachromosomal circle consisting of tandem repeats of plasmid sequences. If multiplication is extensive, the circle forms DMs. If the large circle is integrated into the chromosome, it efficiently initiates a breakage-fusion-bridge cycle, which generates an HSR. Plasmid-borne genes are expressed more actively when they are amplified in DMs than in HSRs (10). Indeed, most of an HSR is heterochromatic, and transcription is detected only at few discrete spots inside the HSR (11). If an inducible promoter in the ...