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.

项目概要/摘要 端粒——定义并保护人类线性染色体的末端——作为一种自然检查 致癌作用。基因组稳定性需要细胞将端粒与危险的 DNA 双链区分开来 断裂（DSB）以阻止不适当的 DSB 修复和 DNA 损伤反应（DDR）信号传导，人类 通过庇护所完成。端粒酶维持性腺和某些茎中端粒的长度 细胞，但体细胞中的端粒随着每次细胞分裂而缩短，这是由于 端粒酶。早期肿瘤中不受限制的细胞分裂最终导致一些端粒变得至关重要 短且激活持续的 DDR 信号传导，导致具有功能性 p53 和 Rb 途径的细胞 经历衰老或凋亡。这些途径有缺陷的细胞继续分裂，直到形成多个端粒 失去保护，然后进入端粒危机，其定义是由于无法耐受的基因组导致细胞活力较差 不稳定，因为染色体在末端反复融合和断裂。临床肿瘤摆脱危机 重新排列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，而端粒酶可以通过以下方式实现这一点 用新端粒直接修复非端粒染色体末端。 拟议项目的目标是识别端粒酶并对其进行机械表征 介导人类细胞中的 DSB 修复。在体外，端粒酶可以将 TTAGGG 重复序列添加到非端粒上 断点序列源自因染色体末端截断而患有 α 地中海贫血的患者。 利用这个序列，我设计了一种基于 PCR 的报告基因检测方法来检测细胞中新端粒的形成 一种诱导型 DSB，并收集了证据表明端粒愈合发生在人类细胞中 端粒酶依赖性方式。我将在 qPCR 平台上使用 TaqMan 探针改进该测定，以严格 量化端粒愈合事件并将进行进一步的实验来证明端粒酶 负责 TTAGGG 重复添加。因为端粒酶介导的修复有可能将 DSB 转化为 末端染色体缺失，我假设人类细胞已经进化出阻断端粒酶的机制 DSB 的活动。我将通过我的端粒愈合测定实施遗传方法来识别生理学 这种 DSB 修复异常模式的抑制因子。最终，我的目标是揭示端粒酶在使 早期癌症跨越端粒危机的瓶颈。我们对基因组理解的飞跃 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。和 在这个奖项的帮助和三机构医学博士/博士项目的激励环境下，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 目标是领导一个以癌症为中心的实验室，同时作为变革性的医生科学家为患者提供护理。