Unusual Telomeric DNAs in Human Telomerase-Negative Immortalized Cells
Unusual Telomeric DNAs in Human Telomerase-Negative Immortalized Cells
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DOI：
10.1128/mcb.00603-08
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发表时间：
2009-02-01
影响因子：
5.3
通讯作者：
Ishikawa, Fuyuki
中科院分区：
文献类型：
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作者：
Nabetani, Akira;Ishikawa, Fuyuki
很大一部分人类癌细胞和永生化细胞以一种不依赖端粒酶的方式维持端粒，这种方式被称为端粒选择性延长（ALT）。有人认为，ALT涉及同源重组，有望产生独特的中间dna。然而，ALT的确切分子机制尚不清楚。为了深入了解端粒dna （t - dna）是如何在ALT中维持的，我们检查了ALT细胞中t - dna的物理结构。我们在t - dna的G链和C链上都发现了丰富的单链区域。此外，二维凝胶电泳和天然凝胶内杂交分析揭示了新的alt特异性单链t- dna，除了之前报道的t-环。这些新发现的alt特异性t- dna包括(i) t复合物，它由具有大量内部单链部分的高度分支t- dna组成；（ii） ss-G，主要由线性单g链t - dna组成；（iii） ss-C，最有可能由环状单c链t - dna组成。用Hirt方法分离细胞dna发现t-circle和ss-G作为染色体外和染色质相关的dna存在于ALT细胞中。我们认为这些ALT特异性t - dna是通过ALT特异性的端粒代谢，即同源重组和滚圈复制机制产生的。
A significant fraction of human cancer cells and immortalized cells maintain telomeres in a telomerase-independent manner called alternative lengthening of telomeres (ALT). It has been suggested that ALT involves homologous recombination that is expected to generate unique intermediate DNAs. However, the precise molecular mechanism of ALT is not known. To gain insight into how telomeric DNAs (T-DNAs) are maintained in ALT, we examined the physical structures of T-DNAs in ALT cells. We found abundant single-stranded regions in both G and C strands of T-DNAs. Moreover, two-dimensional gel electrophoreses and native in-gel hybridization analyses revealed novel ALT-specific single-stranded T-DNAs, in addition to previously reported t-circles. These newly identified ALT-specific T-DNAs include (i) the t-complex, which consists of highly branched T-DNAs with large numbers of internal single-stranded portions; (ii) ss-G, which consists of mostly linear single-G-strand T-DNAs; and (iii) ss-C, which consists of most likely circular single-C-strand T-DNAs. Cellular-DNA fractionation by the Hirt protocol revealed that t-circles and ss-G exist in ALT cells as extrachromosomal and chromatin-associated DNAs. We propose that such ALT-specific T-DNAs are produced by telomere metabolism specific to ALT, namely, homologous recombination and the rolling-circle replication mechanism.