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

项目总结 辐射或接触有毒化学物质造成的端粒损伤可导致癌症并加速 衰老。损伤也可能是正常代谢造成的，包括产生甲醛或转录 当RNA以R-环的形式嵌入DNA时。基因组R-环的广泛研究 表明它们既在转录调控中发挥积极作用，又在导致DNA的有害作用中发挥作用 破裂、突变和癌症。端粒R环(tel-R环)可能是最大的单一来源 端粒的DNA损伤。TEL-R环存在于正常人细胞中，但在细胞中更为丰富 表达ALT癌表型和DNA甲基酶突变的细胞产生高水平的 端粒RNA(Terra)。Tel-R环水平的升高与端粒损伤、缩短和 高重组导致癌症。辐射、顺铂、甲醛等有毒物质，以及暴露于 氧化损伤也可能产生更高水平的tel-R-loop。我们证明了端粒是 排列在大的循环(t-循环)中，最近进行了一项范式转换的发现，将t-循环的形成与 端粒转录产生Terra并产生tel-R-loop，我们认为这是t-loop的关键 队形。因此，TEL-R环是有毒的，也是形成保护性T环所必需的。TELL-R-LOOP更多 由于G-四重奏的形成，比正常的R-环稳定。对基因组和tel-R环的广泛研究已经 所有这些都依赖于使用抗DNA/RNA杂交物的S9.6抗体的单一检测(点滴试验)。在开车的同时 在这个领域，这种IP分析不区分DNA片段上的一个或多个R-环，或者提供 有关R环的聚集或其大小的信息。对于tel-R环路，IP分析不会揭示 在T-环的环部分内有R-环，或者它们从亚端粒区域分布到 端粒末端。为了使这一领域取得进展，必须获得这样的关键信息。现在可以这样做了 使用我们已经验证和正在使用的方法的直接电子显微镜(EM)可视化。 在拟议的工作中，我们将对大(120-240 nT)粒子进行高分辨率研究，这些粒子是由 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环路的频率、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应用一种新型的亲和分离。这件事会做到的 使用培养的HeLa和人ALT癌细胞，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中引入交联剂。一种新的化学基因组定位方法 ROS产生的8-oxoG损伤，特别是在细胞的端粒上，将在合作中应用 已确定对tel-R-环路的影响。T-环结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分解酶切割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.