Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells
Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells
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DOI：
10.1074/jbc.m109.008128
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发表时间：
2009-09-04
影响因子：
4.8
通讯作者：
Shimizu, Noriaki
中科院分区：
文献类型：
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作者：
Harada, Seiyu;Uchida, Masafumi;Shimizu, Noriaki
我们先前发现，携带复制起始区的质粒有效地启动哺乳动物细胞中的基因扩增，并且它产生染色体外双微体和/或染色体均匀染色区。在分析潜在机制的过程中，我们偶然发现发夹帽线性DNA在人类癌细胞系中稳定地维持为大量染色体外微小附加体几个月以上。此类附加体的产生取决于原始质粒中复制起始区的存在。尽管染色体外的维护，附加型基因表达的表观遗传抑制。克隆细胞DNA的Southern印迹分析显示，发夹末端周围的区域在克隆之间是多样化的。此外，亚硫酸氢盐修饰的PCR和测序分析表明，来自原始发夹末端或其末端切除结构的回文序列在克隆长期生长中得到了很好的保留。从这些数据中，我们提出了一个模型，解释这些附加体的形成和维护，其中复制的发夹帽的DNA和十字形的形成及其分辨率发挥核心作用。我们的发现可能与哺乳动物复制子序列的解剖有关。
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.