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

• 批准号: 10569542
• 负责人: JACK D GRIFFITH
• 金额: $ 33.72万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569542
• 关键词: 8-hydroxyguanosine; Acceleration; Affinity; Aging; Antibodies; Binding; Biological Assay; Cells; Chromatin Loop; Chromosomal Rearrangement; Cisplatin; Collaborations; Complex; Cryoelectron Microscopy; DNA; DNA Damage; DNA Sequence; Educational workshop; Electrons; Exposure to; Formaldehyde; Frequencies; Funding; G-Quartets; Generations; Genes; Genetic Recombination; Genetic Transcription; Genomic Instability; Genomics; Hand; Hela Cells; Human; Hybrids; Immunoprecipitation; Laboratories; Lead; Left; Lesion; Link; Location; Malignant Neoplasms; Maps; Measures; Metabolism; Methods; Methyltransferase; Microscopic; Mutagenesis; Mutate; Nucleic Acids; Oxygen; Peer Review; Phenotype; Plasmids; Play; Poison; Preparation; Proteins; Publications; RNA; Radiation; Resolution; Resolvase; Role; Sampling; Single-Stranded DNA; Site; Small Interfering RNA; Source; Structure; System; Technology; Telomerase; Testing; Training; Transcriptional Regulation; United States National Institutes of Health; Visualization; Work; crosslink; in vivo; instrumentation; novel; oxidative damage; particle; protein complex; ribonuclease H1; telomere; tool

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.

项目摘要 辐射或接触有毒化学物质导致的端粒损伤可导致癌症， 衰老正常代谢也可能导致损伤，包括甲醛或转录的产生。 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，也在导致DNA断裂中发挥有害作用。 断裂、突变和癌症。端粒R环可能是最大的来源 端粒DNA损伤Tel-R环存在于正常人类细胞中，但在细胞中更丰富 表达ALT癌症表型的细胞和DNA甲基化酶突变的细胞， 端粒RNA（TERRA）。tel-R环水平的升高与端粒损伤、缩短和 高度重组导致癌症。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-环。我们证明了端粒是 排列成大环（t环），最近做出了一个范式转变的发现，将t环的形成与 端粒转录产生TERRA并产生tel-R环，我们认为tel-R环是t环的关键 阵因此，tel-R环是有毒的，并且是形成保护性t环所必需的。Tel-R-Loop比 由于G-四重体的形成，比正常的R-环稳定。对基因组和tel-R环的广泛研究 所有这些都依赖于使用DNA/RNA杂交体的S9.6抗体的单一试验（DRIP试验）。在开车的时候， 在本领域中，该IP测定不能区分DNA片段上的一个或多个R环，或提供 关于R环的聚类或其大小的信息。对于tel-R-环，IP测定不能揭示 在t-环的环状部分内存在R-环或它们从亚端粒区到端粒区的分布， 端粒末端为了使该领域取得进展，必须获得这些关键信息。现在可以做到这一点 使用直接电子显微镜（EM）可视化，使用我们已经验证和掌握的方法。 在拟议的工作中，我们将进行高分辨率的研究大（120-240 nt）的粒子形成的 富含G的单链端粒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环的频率、位置、大小和聚集， 将应用一种新的亲和分离端粒DNA，结合我们的EM工具电池。为此将 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， 顺铂和甲醛在DNA中引入交联。一种新的化学光基因组学方法 ROS产生的8-oxoG损伤，特别是在端粒的细胞将应用于合作和 测定对tel-R-环的影响。T环连接可能具有重要的功能作用，这将是 使用测定法来检测T环连接被HJ解离酶切割后的端粒延伸。