IFOM-The FIRC Institute of Molecular Oncology, 20139 Milan, Italy.
CNR-Consiglio Nazionale delle Ricerche, Istituto di Genetica Molecolare, 27100 Pavia, Italy.
Int J Mol Sci. 2020 Dec 21;21(24):9774. doi: 10.3390/ijms21249774.
Because of their intrinsic characteristics, telomeres are genomic loci that pose significant problems during the replication of the genome. In particular, it has been observed that telomeres that are maintained in cancer cells by the alternative mechanism of the lengthening of telomeres (ALT) harbor higher levels of replicative stress compared with telomerase-positive cancer cells. R-loops are three-stranded structures formed by a DNA:RNA hybrid and a displaced ssDNA. Emerging evidence suggests that controlling the levels of R-loops at ALT telomeres is critical for telomere maintenance. In fact, on the one hand, they favor telomere recombination, but on the other, they are a source of detrimental replicative stress. DRIP (DNA:RNA immunoprecipitation) is the main technique used for the detection of R-loops, and it is based on the use of the S9.6 antibody, which recognizes preferentially DNA:RNA hybrids in a sequence-independent manner. The detection of DNA:RNA hybrids in repetitive sequences such as telomeres requires some additional precautions as a result of their repetitive nature. Here, we share an optimized protocol for the detection of telomeric DNA:RNA hybrids, and we demonstrate its application in an ALT and in a telomerase-positive cell line. We demonstrate that ALT telomeres bear higher levels of DNA:RNA hybrids, and we propose this method as a reliable way to detect them in telomeres.
由于其固有特性，端粒是基因组复制过程中存在重大问题的基因组位点。特别是，已经观察到，通过端粒延长的替代机制（ALT）在癌细胞中维持的端粒与端粒酶阳性癌细胞相比具有更高水平的复制应激。R 环是由 DNA:RNA 杂交体和移位的单链 DNA 形成的三链结构。新出现的证据表明，控制 ALT 端粒处的 R 环水平对于端粒维持至关重要。事实上，一方面，它们有利于端粒重组，但另一方面，它们是有害复制应激的来源。DRIP（DNA:RNA 免疫沉淀）是检测 R 环的主要技术，它基于使用 S9.6 抗体，该抗体以序列非依赖性的方式优先识别 DNA:RNA 杂交体。由于其重复性质，在重复序列（如端粒）中检测 DNA:RNA 杂交体需要一些额外的预防措施。在这里，我们分享了一种优化的端粒 DNA:RNA 杂交体检测方案，并证明了其在 ALT 和端粒酶阳性细胞系中的应用。我们证明 ALT 端粒具有更高水平的 DNA:RNA 杂交体，并提出该方法是一种在端粒中可靠检测它们的方法。
