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

基本信息

项目摘要

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.
项目摘要/摘要 端粒(定义和保护人类线性染色体的末端)将其视为自然检查 致癌作用。基因组稳定性需要细胞区分端粒与危险DNA双链 断裂(DSB)以阻止不适当的DSB修复和DNA损伤响应(DDR)信号,人类 与庇护所建筑群一起完成。端粒酶在性腺中保持端粒长度和一些茎 细胞,但是由于产生沉默 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批判性 简短而激活的持续DDR信号传导,这会导致具有功能性p53和RB途径的细胞到达 感染或凋亡。这些途径中有缺陷的细胞继续分裂直到多个端粒 被去保护,然后进入端粒危机,这是由于无法忍受的基因组而定义的。 不稳定,因为染色体在其末端反复融合并破裂。临床肿瘤来自危机 重新排列,非整倍体基因组和端粒维护机制。为了摆脱端粒危机,我 预测恶性细胞必须重建其端粒,并且端粒酶可以通过 直接修复非染色体染色体的结尾。 拟议项目的目的是识别和机械表征端粒酶 - 人类细胞中介导的DSB修复。在体外,端粒酶可以在非telo虫中添加ttaggg重复次数 由于末期染色体截断而导致的α-丘脑贫血患者得出的断点序列。 使用此序列,我设计了一个基于PCR的报告基因测定法以检测细胞中的新旋粒形成 可诱导的DSB并收集了证据,表明端粒愈合发生在A中的人类细胞中 端粒酶依赖性方式。我将在QPCR平台上使用Taqman问题来改进这种测定法 量化端粒愈合事件,并将执行进一步的实验以证明端粒酶是 负责ttaggg重复添加。因为端粒酶介导的维修有可能将DSB转换为 末端染色体缺失,我假设人类细胞具有进化的机制来阻止端粒酶 DSB的活动。我将通过端粒治疗测定法实施一种遗传方法,以识别生理 DSB修复这种异常模式的阻遏物。最终,我的目标是揭示远程酶的新角色 初期癌症穿越端粒危机的瓶颈。我们对基因组的理解的飞跃 早期肿瘤发生的不稳定可能导致意外的方法检测和预防患者的癌症。和 该奖项的帮助以及三机构MD/博士计划的刺激环境,我将成长 从科学,医学和专业上以使我能够晋升为长期职业的方式 领导以癌症为中心的实验室的目标,同时为患者护理作为一种变革性的身体科学家。

项目成果

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Charles Gunnar Kinzig其他文献

Charles Gunnar Kinzig的其他文献

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{{ truncateString('Charles Gunnar Kinzig', 18)}}的其他基金

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
  • 财政年份:
    2021
  • 资助金额:
    $ 5.18万
  • 项目类别:

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