R-loops at the telomere as a toxic source of genomic instability

项目概要 辐射或接触有毒化学物质造成的端粒损伤可能导致癌症并加速癌症的发生 老化。正常代谢也可能造成损害，包括甲醛的产生或转录 当 RNA 以 R 环的形式嵌入 DNA 中时。基因组 R 环的广泛研究 已经表明它们在转录调节中发挥积极作用，同时也发挥导致 DNA 的有害作用 断裂、突变和癌症。端粒 R 环（tel-R 环）可能是唯一最大的来源 端粒 DNA 损伤。 Tel-R-loops 存在于正常人类细胞中，但在细胞中更为丰富 表达 ALT 癌症表型和 DNA 甲基化酶突变的细胞产生高水平 端粒RNA（TERRA）。 tel-R-loops 水平升高与端粒损伤、缩短和 高重组导致癌症。辐射、有毒物质（例如顺铂、甲醛）以及接触 氧化损伤也可能产生更高水平的tel-R-loops。我们证明端粒是 排列成大环（t 环），最近做出了一个范式转变的发现，将 t 环形成与 端粒转录产生 TERRA 并产生 tel-R 环，我们认为这是 t 环的关键 形成。因此，tel-R-环既有毒又是形成保护性T-环所必需的。 Tel-R-loops更多 由于 G 四联体的形成，比正常的 R 环稳定。对基因组和 tel-R-loops 的广泛研究已经 所有这些都依赖于使用 DNA/RNA 杂交体 S9.6 抗体的单一测定（DRIP 测定）。开车时 在现场，该 IP 测定不会区分 DNA 片段上的一个或多个 R 环，也不提供 有关 R 环聚类或其大小的信息。对于 tel-R-loops，IP 分析并未揭示是否 在t环的环部分内存在R环或者它们从亚端粒区域到端粒的分布 端粒末端。为了使该领域取得进展，必须获得此类关键信息。现在可以做到这一点 使用我们已经验证并掌握的方法使用直接电子显微镜 (EM) 可视化。 在拟议的工作中，我们将对由以下物质形成的大（120-240 nt）颗粒进行高分辨率研究 单链富含 G 的端粒 DNA 和 TERRA RNA。这对于理解 tel-R- 的结构至关重要 循环并将由 CryoEM 完成。为了确定 tel-R-loops 的频率、位置、大小和集群，我们 将结合我们的一系列 EM 工具，对端粒 DNA 进行新型亲和分离。这将完成 使用培养的 HeLa 和人类 ALT 癌细胞系，并扩展到用有毒化学物质处理的细胞，包括 顺铂和甲醛在 DNA 中引入交联。一种新的化学光基因组方法 ROS 在细胞端粒处产生 8-oxoG 损伤，将在合作中应用 已确定对 tel-R-loops 的影响。 T 环连接可能具有重要的功能作用，这将是 探索了使用检测方法来检测 HJ 解离酶裂解 T 环连接后的端粒延伸。

PROJECT SUMMARY Damage to telomeres resulting from radiation or exposure to toxic chemicals can lead to cancer and accelerated aging. Damage may also result from normal metabolism including generation of formaldehyde or transcription when RNA is left behind embedded in the DNA in the form of R-loops. Extensive studies of genomic R-loops have shown them to play both positive roles in regulation of transcription and harmful roles leading to DNA breakage, mutagenesis and cancer. Telomeric R-loops (tel-R-loops) may possibly be the single greatest source of DNA damage at telomeres. Tel-R-loops occur in normal human cells, but are more abundant in cells expressing the ALT cancer phenotype and cells mutated in DNA methylases that produce high levels of telomeric RNA (TERRA). Elevated levels of tel-R-loops have been linked to telomere damage, shortening and high recombination leading to cancer. Radiation, toxic agents such as cisplatin, formaldehyde, and exposure to oxidative damage are also likely to generate higher levels of tel-R-loops. We demonstrated that telomeres are arranged in large loops (t-loops) and recently made a paradigm-shifting discovery linking t-loop formation to telomere transcription which generates TERRA and produces tel-R-loops which we propose are key to t-loop formation. Thus, tel-R-loops are both toxic and necessary for forming protective t-loops. Tel-R-loops are more stable than normal R-loops due to G-quartet formation. The extensive studies of genomic and tel-R-loops have all relied on a single assay employing the S9.6 antibody to DNA/RNA hybrids (DRIP assay). While having driven the field, this IP assay does not discriminate between one or many R-loops on a DNA fragment, or provide information on the clustering of the R-loops, or their size. For tel-R-loops, the IP assay does not reveal whether there are R-loops within the looped portion of the t-loop or their distribution from the sub-telomeric regions to the telomere terminus. For the field to progress, such critical information must be obtained. This can now be done using direct electron microscopic (EM) visualization using methods we have verified and in hand. In the proposed work we will carry out a high resolution study of the large (120-240 nt) particles formed by single stranded G-rich telomeric DNA and TERRA RNA. This is critical for understanding the structure of tel-R- loops and will be done by cryoEM. To determine the frequency, location, size and clustering of tel-R-loops we will apply a novel affinity isolation for telomeric DNA, combined with our battery of EM tools. This will be done using cultured HeLa and human ALT cancer lines and extended to cells treated with toxic chemicals including cisplatin and formaldehyde to introduce crosslinks in the DNA. A novel chemoptogenomic approach for placing ROS generated 8-oxoG lesions specifically at the telomere in cells will be applied in a collaboration and the effect on tel-R-loops determined. The t-loop junction may have important functional roles and this will be explored using assays to detect telomere extension following cleavage of the t-loop junction by HJ resolvases.