Single-molecule analysis of subtelomeres and telomeres in Alternative Lengthening of Telomeres (ALT) cells
Single-molecule analysis of subtelomeres and telomeres in Alternative Lengthening of Telomeres (ALT) cells
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DOI：
10.1186/s12864-020-06901-7
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发表时间：
2020-07-15
期刊：
影响因子：
4.4
通讯作者：
Xiao, Ming
中科院分区：
文献类型：
--
作者：
Abid, Heba Z.;McCaffrey, Jennifer;Xiao, Ming
背景端粒DNA通常由富含G的串联重复基序组成，并由端粒酶维持（Greider CW，Blackburn EH; Cell 51：887-898; 1987）。在缺乏端粒酶的真核生物中，在通常依赖端粒酶进行端粒延长的生物体中进化出了多种基于DNA修复和DNA重组的端粒维持途径（Webb CJ，Wu Y，Zakian VA; Cold Spring Harb Perspect Biol 5：a012666; 2013）;统称为端粒替代延长（ALT）途径。通过测量（TTAGGG）n道长度从相同的大DNA分子的光学映射，我们同时分析了端粒长度动态和亚端粒连接的结构变化在大量的特定亚端粒位点的ALT阳性细胞系U2 OS，SK-MEL-2和Saos-2。结果ALT端粒具有位点特异性。例如，虽然每个亚端粒包括具有末端（TTAGGG）n束的单分子的例子以及重组端粒单分子的例子，但这些分子的比例是亚端粒特异性的，在U2 OS中范围从33：1（19 p）到1：25（19 q）。Saos-2细胞系显示出类似的重组端粒百分比。SK-MEL-2的重组亚端粒的频率（11%）约为U2 OS和Saos-2的一半（分别为24%和19%）。末端（TTAGGG）n道长度和异质性水平，端粒信号自由端的频率，和保留的内部端粒样序列（ITS）在重组端粒融合交界处的频率和大小都不同，根据具体的亚端粒在一个特定的细胞系。在所有三种细胞系中均发现了非常大的线性染色体外端粒重复序列（ECTR）DNA分子;这些分子原则上能够通过断裂诱导修复（BIR）长链DNA合成机制模板合成新的长端粒链，并有助于ALT细胞的非常长的端粒链长度和异质性特征。许多最长的端粒束（末端端粒和线性ECTR）显示点状CRISPR/Cas9依赖性（TTAGGG）n标记模式，指示非典型端粒重复序列的延伸的散布。结论利用我们的新的单分子方法鉴定单个亚端粒并表征连接端粒（TTAGGG）n束长度和结构变化，以前所未有的分子细节和不同ALT阳性细胞系的显著差异揭示了人ALT细胞中端粒损伤、修复和重组机制的结构后果。
Background Telomeric DNA is typically comprised of G-rich tandem repeat motifs and maintained by telomerase (Greider CW, Blackburn EH; Cell 51:887-898; 1987). In eukaryotes lacking telomerase, a variety of DNA repair and DNA recombination based pathways for telomere maintenance have evolved in organisms normally dependent upon telomerase for telomere elongation (Webb CJ, Wu Y, Zakian VA; Cold Spring Harb Perspect Biol 5:a012666; 2013); collectively called Alternative Lengthening of Telomeres (ALT) pathways. By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2. Results Our results revealed loci-specific ALT telomere features. For example, while each subtelomere included examples of single molecules with terminal (TTAGGG) n tracts as well as examples of recombinant telomeric single molecules, the ratio of these molecules was subtelomere-specific, ranging from 33:1 (19p) to 1:25 (19q) in U2OS. The Saos-2 cell line shows a similar percentage of recombinant telomeres. The frequency of recombinant subtelomeres of SK-MEL-2 (11%) is about half that of U2OS and Saos-2 (24 and 19% respectively). Terminal (TTAGGG) n tract lengths and heterogeneity levels, the frequencies of telomere signal-free ends, and the frequency and size of retained internal telomere-like sequences (ITSs) at recombinant telomere fusion junctions all varied according to the specific subtelomere involved in a particular cell line. Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were found in all three cell lines; these are in principle capable of templating synthesis of new long telomere tracts via break-induced repair (BIR) long-tract DNA synthesis mechanisms and contributing to the very long telomere tract length and heterogeneity characteristic of ALT cells. Many of longest telomere tracts (both end-telomeres and linear ECTRs) displayed punctate CRISPR/Cas9-dependent (TTAGGG) n labeling patterns indicative of interspersion of stretches of non-canonical telomere repeats. Conclusion Identifying individual subtelomeres and characterizing linked telomere (TTAGGG) n tract lengths and structural changes using our new single-molecule methodologies reveals the structural consequences of telomere damage, repair and recombination mechanisms in human ALT cells in unprecedented molecular detail and significant differences in different ALT-positive cell lines.