title: 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.
authors.affiliation: Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima 739-8521, Japan.
authors.forename: Seiyu
authors.initials: S
authors.lastname: Harada
authors.forename: Masafumi
authors.initials: M
authors.lastname: Uchida
authors.forename: Noriaki
authors.initials: N
authors.lastname: Shimizu
mesh_terms: D045744:Cell Line, Tumor; D004261:DNA Replication / Q000502:physiology*; D005784:Gene Amplification / Q000502:physiology*; D006801:Humans; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin / Q000502:physiology*