Structural and Biological Effects of Ribonucleotide Insertion into Telomeres

核糖核苷酸插入端粒的结构和生物学效应

• 批准号: 10750783
• 负责人: Luis Manuel Cortez
• 金额: $ 3.59万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750783
• 关键词: Address; Affect; Antibodies; Apoptosis; Biochemical; Biological; Biological Assay; Biophysics; Biotin; Cancer Biology; Cell Culture Techniques; Cell Line; Cells; Cellular Assay; Chromosomes; Circular Dichroism; Circular Dichroism Spectroscopy; Complement; Critical Thinking; DNA; DNA Damage; DNA Folding; DNA annealing; DNA biosynthesis; Data; Doctor of Philosophy; Ensure; Enzymes; Excision Repair; G-Quartets; Genome; Genomic Instability; Goals; Higher Order Chromatin Structure; In Vitro; Left; Length; Lesion; Link; Maintenance; Malignant Neoplasms; Mechanics; Molecular Conformation; Monitor; Nucleotides; Oligonucleotides; Pathway interactions; Physicians; Proteins; RNA; RNA-Directed DNA Polymerase; Repetitive Sequence; Research; Ribonucleotides; Scientist; Spectrophotometry; Structure; System; Telomerase; Testing; Tissues; Training; Transfection; Untranslated RNA; career; experimental study; genome integrity; human disease; innovation; mutant; reconstitution; repaired; senescence; skills; telomere

Project Abstract Telomeres are protective noncoding DNA caps at the ends of chromosomes that maintain genomic integrity. In most somatic tissues telomeres will shorten with successive rounds of replication and will reach a critically short length, at which point a cell will become senescent or undergo apoptosis. This fate can be avoided if telomeres are maintained through expression of telomerase, a reverse transcriptase that elongates telomeres by adding telomeric repeats. This elongation of telomeres can lead to replicative immortality, one of the hallmarks of cancer. Another hallmark of cancer is genomic instability that arises from DNA damage. The most prevalent form of DNA damage are ribonucleotides (rNTPs) inserted during DNA replication. Left unrepaired these rNTPs will promote genomic instability, changes to the DNA secondary structure, and human diseases. Given the deleterious effects of rNTPs in DNA, cells have evolved the ribonucleotide excision repair (RER) pathway to remove rNTPs. While the impact of rNTPs in the genome is well established to have deleterious effects and promote human disease, the impact of rNTPs at telomeres remains unknown. One essential DNA secondary structure seen at telomeres, that can be affected by rNTPs, is a G-quadruplex (G4). Specific to this proposal, it is not known what effect rNTPs will have on telomeric structure and integrity, or how rNTPs at telomeres are repaired to protect telomere integrity. To investigate this, we have developed a telomerase mutant (Y717A) that inserts rNTPs at a greater rate than wildtype (WT) telomerase. Using this mutant, I will selectively increase the rate of rNTP incorporation only at telomeres. The overarching goal of this proposal is to determine the effect of rNTPs on telomeric G4s and characterize the repair pathway for rNTPs in telomeres. I hypothesize that rNTP insertion into telomeres will alter telomeric G4 dynamics and that these rNTPs are repaired through RER. To test this hypothesis, I propose the following specific aims: 1) characterize the structural effects of rNTP insertion into telomeres and 2) characterize rNTP repair in telomeres. For aim 1, I will use circular dichroism spectrophotometry to systematically examine G4 formation in vitro. Using oligos of the basic telomeric sequence that will form a G4 (TTAGGG)4, I will systematically replace the dNTPs with rNTPs to determine if rNTPs alter the key G4 structural motif in telomeres. Additionally, using cell lines that contain either WT telomerase or a telomerase mutant which inserts rNTPs at a greater rate than WT in conjunction with antibodies that are specific for G4s to examine if the presence of rNTPs alters the G4 content at telomeres. For aim 2, I will characterize RER by reconstituting the RER pathway with a substrate that has a single rNTP that will either be linear DNA or DNA folded into a G4. I will monitor the formation of the repaired product and compare RER functionality on the different substrates. Additionally, using proximity dependent biotin identification (BioID) I will be able to identify any additional proteins that are involved in repair of rNTPs at telomeres. This study applies an innovative combination of in vitro biochemical assays with cellular assays to examine the impact and repair of rNTPs at telomeres.

项目摘要 端粒是染色体末端的保护性非编码 DNA 帽，可维持基因组完整性。在 大多数体细胞组织的端粒会随着连续几轮的复制而缩短，并达到极短的程度。 长度，此时细胞将衰老或发生凋亡。如果端粒能够避免这种命运 通过端粒酶的表达来维持，端粒酶是一种逆转录酶，通过添加延长端粒 端粒重复。端粒的延长可以导致复制永生，这是癌症的标志之一。 癌症的另一个标志是 DNA 损伤引起的基因组不稳定。 DNA最普遍的形式 损伤是 DNA 复制过程中插入的核糖核苷酸 (rNTP)。如果不修复这些 rNTP 将促进 基因组不稳定性、DNA 二级结构的变化和人类疾病。鉴于有害影响 DNA 中 rNTP 的存在，细胞已经进化出核糖核苷酸切除修复 (RER) 途径来去除 rNTP。尽管 rNTP 对基因组的影响已被证实会产生有害影响并促进人类疾病， rNTP 对端粒的影响仍不清楚。在端粒上看到的一种重要的 DNA 二级结构， 可以受 rNTP 影响的是 G-四联体 (G4)。具体到这个提案，还不知道有什么效果 rNTP 将影响端粒结构和完整性，或者端粒上的 rNTP 如何修复以保护端粒 正直。为了研究这一点，我们开发了一种端粒酶突变体 (Y717A)，它可以以更高的频率插入 rNTP。 速率高于野生型 (WT) 端粒酶。使用这个突变体，我将选择性地提高 rNTP 掺入率 仅在端粒处。该提案的总体目标是确定 rNTP 对端粒 G4 的影响 并表征端粒中 rNTP 的修复途径。我假设 rNTP 插入端粒会 改变端粒 G4 动力学，并且这些 rNTP 通过 RER 进行修复。为了检验这个假设，我建议 以下具体目标：1) 表征 rNTP 插入端粒的结构效应；2) 表征端粒中的 rNTP 修复。对于目标 1，我将使用圆二色分光光度法来系统地 体外检查 G4 形成。使用基本端粒序列的寡核苷酸形成 G4 (TTAGGG)4，我 将系统地用 rNTP 替换 dNTP，以确定 rNTP 是否改变了关键的 G4 结构基序 端粒。此外，使用含有 WT 端粒酶或插入端粒酶突变体的细胞系 rNTP 的速率高于 WT 与 G4 特异性抗体的结合，以检查是否存在 rNTPs 改变端粒上的 G4 含量。对于目标 2，我将通过重构 RER 路径来表征 RER 底物具有单个 rNTP，该 rNTP 可以是线性 DNA 或折叠成 G4 的 DNA。我会监控 修复产物的形成并比较不同基材上的 RER 功能。此外，使用 邻近依赖性生物素识别 (BioID) 我将能够识别所涉及的任何其他蛋白质 修复端粒上的 rNTP。这项研究应用了体外生化检测的创新组合 细胞检测来检查 rNTP 对端粒的影响和修复。