Telomerase-Mediated Healing of Double-Strand Breaks in Human Cells

端粒酶介导的人体细胞双链断裂修复

• 批准号: 10560471
• 负责人: Charles Gunnar Kinzig
• 金额: $ 5.27万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10560471
• 关键词: Aneuploidy; Apoptosis; Award; Base Pairing; Binding; Biological Assay; Cell Aging; Cell Survival; Cell division; Cells; Centromere; Chromosomal Breaks; Chromosome 16; Chromosome Deletion; Chromosomes; Clinical; Complex; DNA Damage; DNA Double Strand Break; DNA Primase; Data; Development; Dicentric chromosome; Distal; Doctor of Philosophy; Double Strand Break Repair; Doxycycline; Ectopic Expression; Environment; Event; Frequencies; Genes; Genome; Genome Stability; Genomic Instability; Genomics; Germ Cells; Goals; Gonadal structure; Haploidy; Hela Cells; Human; In Vitro; Institution; Length; Link; Malignant Neoplasms; Mediating; Medical; Methods; Molecular; Neoplasms; Pathologic; Pathway interactions; Patient Care; Patients; Physicians; Physiological; Polymerase; Process; Proliferating; Regulation; Reporter; Repression; Role; Running; Rupture; Scientist; Signal Transduction; Somatic Cell; TP53 gene; Telomerase; Telomerase Inhibitor; Telomerase RNA Component; Telomerase inhibition; Telomere Maintenance; Telomere Shortening; Testing; Trees; alpha-Thalassemia; cancer cell; cancer diagnosis; cancer prevention; carcinogenesis; career; cell transformation; chromosome fusion; chromothripsis; design; detection assay; expectation; experimental study; genetic approach; healing; improved; mortality; neoplastic cell; novel strategies; p53-binding protein 1; prevent; programs; reconstitution; recruit; repaired; response; senescence; stem cells; telomere; tumor; tumorigenesis

PROJECT SUMMARY/ABSTRACT Telomeres—which define and protect the ends of humans’ linear chromosomes—serve as a natural check on carcinogenesis. Genome stability requires cells to differentiate telomeres from perilous DNA double-strand breaks (DSBs) to block inappropriate DSB repair and DNA damage response (DDR) signaling, which humans accomplish with the shelterin complex. Telomerase maintains telomere length in the gonads and some stem cells, but telomeres in somatic cells shorten with each cell division due to developmental silencing of telomerase. Unfettered cell division in early neoplasms eventually leads to a few telomeres becoming critically short and activating persistent DDR signaling, which causes cells with functional p53 and Rb pathways to undergo senescence or apoptosis. Cells defective in these pathways continue to divide until multiple telomeres become de-protected and then enter telomere crisis, defined by poor cell viability due to intolerable genomic instability, as chromosomes repeatedly fuse at their ends and break. Clinical tumors emerge from crisis with rearranged, aneuploid genomes and a telomere maintenance mechanism. To escape from telomere crisis, I predict that malignant cells must reconstitute their telomeres and that telomerase may accomplish this by directly repairing non-telomeric chromosome ends with neotelomeres. The objective of the proposed project is to identify and mechanistically characterize telomerase- mediated DSB repair in human cells. In vitro, telomerase can add TTAGGG repeats to a non-telomeric breakpoint sequence derived from a patient with α-thalassemia due to a terminal chromosomal truncation. Using this sequence, I have designed a PCR-based reporter assay to detect neotelomere formation in cells at an inducible DSB and have gathered evidence that suggests that telomere healing occurs in human cells in a telomerase-dependent manner. I will improve this assay with TaqMan probes on a qPCR platform to rigorously quantify telomere healing events and will perform further experiments to demonstrate that telomerase is responsible for TTAGGG repeat addition. Because telomerase-mediated repair threatens to convert DSBs into terminal chromosome deletions, I hypothesize that human cells have evolved mechanisms to block telomerase activity at DSBs. I will implement a genetic approach with my telomere healing assay to identify the physiologic repressors of this aberrant mode of DSB repair. Ultimately, I aim to unveil a new role for telomerase in enabling incipient cancers to traverse the bottleneck of telomere crisis. This leap in our understanding of genomic instability in early tumorigenesis may lead to unexpected ways to detect and prevent cancer in patients. With the aid of this award and the stimulating environment of the Tri-Institutional MD/PhD Program, I will grow scientifically, medically, and professionally in ways that will enable me to advance toward my long-term career goal of leading a cancer-centric lab while providing patient care as a transformative physician-scientist.

项目总结/摘要 端粒--它定义并保护人类线性染色体的末端--作为一种自然的检查， 致癌作用基因组稳定性要求细胞区分端粒和危险的DNA双链 DNA断裂（DSB）阻断不适当的DSB修复和DNA损伤反应（DDR）信号， 完成与shelterin复杂。端粒酶在性腺和某些茎中维持端粒长度 细胞，但体细胞中的端粒缩短与每个细胞分裂由于发育沉默， 端粒酶早期肿瘤中不受约束的细胞分裂最终导致少数端粒变得严重 缩短并激活持续的DDR信号传导，导致具有功能性p53和Rb通路的细胞 经历衰老或凋亡。在这些通路中有缺陷的细胞继续分裂，直到多个端粒 变得去保护，然后进入端粒危机，其定义为由于不可耐受的基因组DNA而导致的细胞活力差。 不稳定性，因为染色体在其末端反复融合并断裂。临床肿瘤摆脱危机， 重排的非整倍体基因组和端粒维持机制。为了摆脱端粒危机，我 预测恶性细胞必须重建它们的端粒，而端粒酶可能通过以下方式完成这一任务： 用新端粒直接修复非端粒染色体末端。 该项目的目标是识别和机械表征端粒酶- 介导的DSB修复。在体外，端粒酶可以将TTAGGG重复序列添加到非端粒序列， 由于末端染色体截短而衍生自患有α-地中海贫血的患者的断裂点序列。 利用这个序列，我设计了一个基于PCR的报告分析，以检测细胞中新端粒的形成， 一种可诱导的DSB，并收集了证据表明，端粒愈合发生在人类细胞中， 端粒酶依赖的方式。我将在qPCR平台上使用TaqMan探针改进该检测方法，以严格 量化端粒愈合事件，并将进行进一步的实验，以证明端粒酶是 负责TTAGGG重复添加。因为端粒酶介导的修复威胁着将DSB转化为 由于染色体末端缺失，我假设人类细胞已经进化出了阻断端粒酶的机制 在DSB的活动。我将用我的端粒愈合试验实施一种遗传方法， 这种异常的DSB修复模式的阻遏物。最终，我的目标是揭示端粒酶的新作用， 通过端粒危机的瓶颈。我们对基因组的理解 早期肿瘤发生的不稳定性可能会导致意想不到的方法来检测和预防患者的癌症。与 这个奖项的援助和三机构的MD/博士课程的刺激环境，我会成长 科学、医学和专业的方式使我能够朝着我的长期职业生涯前进 领导一个以癌症为中心的实验室，同时作为一个变革性的医生科学家提供病人护理的目标。