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

端粒上的 R 环是基因组不稳定的毒源

基本信息

批准号：
10770896
负责人：
JACK D GRIFFITH
金额：
$ 2.13万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-21 至 2024-01-31
项目状态：
已结题

项目摘要

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.

抽象的辐射或接触有毒化学物质造成的端粒损伤可能导致癌症和由于端粒缩短而加速衰老。正常过程也可能造成损坏包括当 RNA 被留在 DNA 中时产生甲醛或转录 R 环的形式。对基因组 R 环的广泛研究表明它们在转录调控和导致 DNA 断裂和突变的有害作用。端粒 R 环 (t- R-环）可能是端粒 DNA 损伤的最大单一来源。 T-R 环正常发生人类细胞中 ALT 含量更高，癌细胞和某些 DNA 甲基化酶突变的细胞导致高水平的端粒RNA (TERRA)。 t-R 环水平升高与端粒有关损坏和缩短。辐射、有毒物质（如顺铂、甲醛）和暴露于氧化环境损伤也可能产生更高水平的 t-R 环。我们证明端粒排列在大环（t 环），最近做出了将 t 环形成与端粒联系起来的范式转变发现生成 TERRA 并产生 t-R 环的转录，我们认为这是 t 环形成的关键。因此，端粒 R 环可能具有毒性，并且是形成保护性 T 环所必需的。 T-R环更多由于 G 四联体的形成，比正常的 R 环稳定。基因组和端粒 R 的广泛研究环全部依赖于使用针对 DNA/RNA 杂交体的高度特异性 S9.6 抗体 (DRIP 测定）。在推动该领域发展的同时，该 IP 测定不会区分同一样品上的一个或多个 R 环。 DNA 片段或提供有关 R 环聚类或其大小的信息。对于 t-R 环，IP 分析没有揭示 t 环的环部分内是否存在 R 环或其分布亚端粒区域至端粒末端。为了使该领域取得进展，这些关键信息必须获得。现在可以使用我们拥有的方法通过直接电子显微镜 (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，我们对由单链富含 G 的端粒 DNA 形成的大（120-240 nt）颗粒，我们提出 TERRA RNA。这对于理解 t-R 环的结构至关重要，并将通过冷冻电镜完成。直接确定 t-R-环的频率、位置、大小和聚类，我们将为端粒 DNA 应用新型亲和分离，与一组 EM 工具相结合。这将使用培养的 HeLa 和人类 ALT 癌细胞系来完成扩展到用有毒化学物质（包括顺铂和甲醛）处理的细胞，以在细胞中引入交联 DNA。一种新的化学光基因组方法，用于将 ROS 产生的 8-oxo-G 病变特异性放置在细胞中的端粒将在合作中应用，并确定 t-R 环水平的结果。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The mitochondrial single-stranded DNA binding protein is essential for initiation of mtDNA replication.

线粒体单链 DNA 结合蛋白对于 mtDNA 复制的启动至关重要。

DOI：
发表时间：
2021
期刊：
影响因子：
13.6
作者：
Jiang, Min;Xie, Xie;Zhu, Xuefeng;Jiang, Shan;Milenkovic, Dusanka;Misic, Jelena;Shi, Yonghong;Tandukar, Nirwan;Li, Xinping;Atanassov, Ilian;Jenninger, Louise;Hoberg, Emily;Albarran;Szilagyi, Zsolt;Macao, Bertil;Siira, Stefan
通讯作者：
Siira, Stefan

Mammalian telomeric RNA (TERRA) can be translated to produce valine-arginine and glycine-leucine dipeptide repeat proteins.

哺乳动物端粒 RNA (TERRA) 可被翻译产生缬氨酸-精氨酸和甘氨酸-亮氨酸二肽重复蛋白。