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

摘要 辐射或接触有毒化学物质导致的端粒损伤可导致癌症， 由于不必要的端粒缩短而加速老化。正常过程也可能导致损坏 包括甲醛的产生或转录，当RNA被留在DNA中时， R-loop的形式。对基因组R环的广泛研究表明，它们在基因组中既发挥积极作用， 转录调控和有害作用导致DNA断裂和诱变。端粒R环（t- R环）可能是端粒DNA损伤的最大来源。T-R环发生在正常的 在人细胞中更丰富，在ALT癌细胞和某些DNA甲基化酶突变的细胞中更丰富， 端粒RNA（TERRA）的高水平。t-R环水平的升高与端粒有关 损伤和缩短。辐射、有毒物质（如顺铂、甲醛）和暴露于氧化剂 损伤也可能产生更高水平的t-R环。我们证明了端粒是以 大环（T环），最近作出了一个范式转变的发现，连接T环的形成端粒 转录产生TERRA和产生t-R环，我们认为这是t环形成的关键。 因此，端粒R环可能是有毒的，也是形成保护性T环所必需的。T-R循环更像是 由于G-四重体的形成，比正常的R-环稳定。基因组和端粒R- 循环都依赖于一种单一的检测方法，该方法采用了针对DNA/RNA杂交体的高度特异性S9.6抗体（DRIP 测定）。虽然已经驱动了场，但是该IP测定法不能区分一个或多个R环上的R环。 DNA片段或提供关于R环的聚类或其大小的信息。对于t-R环，IP测定 没有揭示T环的环状部分内是否有R环或它们在T环中的分布。 亚端粒区到端粒末端。为了使该领域取得进展，必须 得到了这现在可以使用直接电子显微镜（EM）可视化来完成， 在基于质粒的R环研究中得到验证。在我们提出的工作中，我们进行了高分辨率的研究， 大（120-240 nt）颗粒形成的单链G丰富的端粒DNA，我们建议，TERRA RNA。 这对于理解t-R环的结构至关重要，将通过cryoEM进行。直接确定 t-R环的频率、位置、大小和聚类，我们将应用一种新的端粒DNA亲和分离， 结合一系列电磁工具这将使用培养的HeLa和人ALT癌细胞系进行， 扩展到用包括顺铂和甲醛在内的有毒化学物质处理的细胞， DNA一种新的化学光基因组学方法，用于将ROS产生的8-oxo-G损伤特异性地放置在 端粒在细胞中将被应用在一个合作和结果的水平上的t-R环确定。

Abstract Damage to telomeres resulting from radiation or exposure to toxic chemicals can lead to cancer and accelerated aging due to unwanted telomere shortening. Damage may also result from normal processes 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 and mutagenesis. Telomeric R-loops (t- R-loops) may possibly be the single greatest source of DNA damage at telomeres. T-R-loops occur in normal human cells and are more abundant in ALT cancer cells and cells mutated in certain DNA methylases that result in high levels of telomeric RNA (TERRA). Elevated levels of t-R-loops have been linked to telomere damage and shortening. Radiation, toxic agents such as cisplatin, formaldehyde, and exposure to oxidative damage are also likely to generate higher levels of t-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 t-R-loops which we propose are key to t-loop formation. Thus, telomeric R-loops may be both toxic and necessary for forming protective t-loops. T-R-loops are more stable than normal R-loops due to G-quartet formation. The extensive studies of genomic and telomeric R- loops have all relied on a single assay employing the highly specific 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 t-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 in a plasmid-based R-loop study. In our proposed work we carry out a high-resolution study of the large (120-240 nt) particles formed by single stranded G-rich telomeric DNA and we propose, TERRA RNA. This is critical for understanding the structure of t-R-loops and will be done cryoEM. To directly determine the frequency, location, size and clustering of t-R-loops we will apply a novel affinity isolation for telomeric DNA, combined with a 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-oxo-G lesions specifically at the telomere in cells will be applied in a collaboration and the result on the levels of t-R-loops determined.