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 端粒是维持基因组完整性的染色体末端的保护性非编码DNA帽。在 大多数体细胞组织的端粒会因连续的复制而缩短，并且至关重要 长度，这一点，一个细胞将变成衰老或凋亡。如果端粒可以避免这种命运 通过表达端粒酶来维护，端粒酶是一种逆转录酶，通过添加来拉长端粒 端粒重复。端粒的这种伸长可能导致复制不朽，这是癌症的标志之一。 癌症的另一个标志是基因组不稳定性是由DNA损伤引起的。 DNA最普遍的形式 损伤是在DNA复制过程中插入的核糖核苷酸（RNTP）。这些RNTP会促进未修复 基因组不稳定性，DNA二级结构的变化和人类疾病。鉴于有害效果 DNA中的RNTPS的细胞已进化为核糖核苷酸切除修复（RER）途径以去除RNTP。尽管 RNTP在基因组中的影响已经良好，可以具有有害作用并促进人类疾病， RNTP对端粒的影响仍然未知。在端粒中看到的一个必需的DNA二级结构， RNTP的影响是G Quadruplex（G4）。特定于该提议，尚不知道什么效果 RNTPS将具有端粒结构和完整性，或如何修复端粒的RNTP来保护端粒 正直。为了调查这一点，我们开发了一个端粒酶突变体（Y717A），该突变体将RNTP插入更大 比野生型（WT）端粒酶的速率。使用此突变体，我将有选择地提高RNTP掺入速率 仅在端粒。该提案的总体目标是确定RNTP对端粒G4S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，并通过RER修复这些RNTP。为了检验这一假设，我提出了 以下具体目的：1）表征RNTP插入端粒的结构效应，而2） 表征端粒中的RNTP修复。对于AIM 1，我将使用圆形二分法分光光度法来系统地 在体外检查G4形成。使用将形成G4（ttaggg）4的基本端粒序列的寡聚物，i 将使用RNTP系统地替换DNTP，以确定RNTP是否会改变密钥G4结构图案 端粒。此外，使用包含WT端粒酶或端粒酶突变体的细胞系 RNTP的速率高于WT，与G4s特有的抗体结合使用，以检查是否存在 RNTPS的含量改变了端粒的G4含量。对于AIM 2，我将通过重新建立RER路径来表征RER 具有单个RNTP的底物，该RNTP将是线性DNA或DNA折叠成G4的底物。我将监视 形成修复的产品并比较不同底物上的RER功能。另外，使用 接近性依赖性生物素鉴定（BiOid）我将能够识别任何其他蛋白质 在修复端粒的RNTP中。这项研究采用了体外生化测定的创新组合与 细胞测定以检查端粒RNTP的影响和修复。