Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells

Abstract

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 ...

Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells

“…The transfected sequence was detected as a bright signal at extrachromosomal DMs, the extrachromosomal tiny elements (ETEs) of smaller signals [20], or chromosomal HSRs of varying length (Fig 3A). The frequency of these signals is shown in Fig 3B and 3C.…”

“…It was constructed by cutting the multiple cloning site of plasmid pTV-MCS with Bam HI [ 11 ], followed by insertion of the d2EGFP expression cassette as described above. Construction of pG5 ( Fig 1C ) [ 11 ] and pΔBM d2EGFP ( Fig 1D ) [ 20 ] has been described previously.…”

Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells

“…An IR/MAR plasmid bearing a sequence homologous to the MAC was constructed and is referred to as ‘plasmid 6’ (Figure 1B). The pΔBM.d2EGFP vector (27) harboring an IR/MAR from the Chinese hamster dihydrofolate reductase ( DHFR ) locus, blasticidin resistance ( bsr ) gene expression cassette and intracellular long-lived decay 2 enhanced green fluorescent protein ( d2EGFP ) expression cassette was used as the starting plasmid. We PCR-amplified a 2844 bp sequence in the homologous region (HR)2 sequence (Supplementary Figure S4) located on the MAC (Supplementary Figure S2) using a primer set with a 20-nt HR2-specific sequence linked at the 5′-end to a 15-nt vector cloning site sequence.…”

Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing

“…The construction of pΔBM-d2EGFP was described previously [ 19 ]. This plasmid contains a 4.6 kb IR sequence from a non-coding region of the DHFR locus, including a sequence that exhibits in vitro MAR activity [ 4 ].…”