Project 2 Symington

项目2赛明顿

• 批准号: 10614962
• 负责人: Lorraine S Symington
• 金额: $ 35.23万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-08 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10614962
• 关键词: ANGPTL2 gene; Address; Aneuploidy; BRCA1 gene; Biological Assay; CRISPR/Cas technology; Cell Survival; Cells; Chromosomal Breaks; Chromosomal Rearrangement; Chromosomal translocation; Chromosomes; Collaborations; Complex; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA Sequence; DNA biosynthesis; DNA damage checkpoint; DNA replication fork; Defect; Double Strand Break Repair; Event; Excision; Exhibits; Frequencies; Functional disorder; Gene Family; Genes; Genetic; Genetic Screening; Genome; Genomic Instability; Human; Individual; Knowledge; Left; Lesion; Malignant Neoplasms; Mammary Neoplasms; Measures; Mediating; Methods; Mutagenesis; Mutation; Nature; Nonhomologous DNA End Joining; Outcome; Pathology; Pathway interactions; Process; Proteins; Replication Error; Resected; Role; Signal Pathway; Site; Source; System; Technology; Yeasts; base; cancer cell; cancer genome; clastogen; driving force; endonuclease; experimental study; insertion/deletion mutation; mutant; next generation sequencing; novel; nuclease; prevent; repaired; replication stress; response; telomere; tumor; tumor DNA; tumorigenic

SUMMARY Most human cancer cells exhibit genome instability, ranging from elevated mutation rates (base substitutions and indels) to chromosomal rearrangements (CRs) and aneuploidy. The application of next generation sequencing (NGS) technologies to analyze cancer genomes has resulted in a wealth of information on the spectrum of genomic instability, and led to the identification of specific mutation and CR signatures associated with loss of different DNA repair pathways. The prevailing view is that CRs are generated through error-prone processing of damaged chromosomes. Although the nature of the initiating lesions for CRs is unknown, much of the genetic evidence from yeast and human cells implicates DNA replication errors as a source of the broken chromosomes that fuel CRs. The two aims outlined in this proposal will address the source of initiating lesions for CRs, and measure the frequency and spectra of CRs in wild type and repair-deficient cells in response to defined DNA damage. In the first aim, we will induce site-specific DSBs using CRISPR-Cas9 in yeast or human cells and determine the full spectrum of CRs in surviving cells. The experiments will be performed in cells lacking specific components of non-homologous end joining (NHEJ), homology-dependent repair (HR) or DNA damage signaling pathways to determine how distinct types of CRs are suppressed. We expect to define unique CR signatures for each deficiency that could guide identification of novel CR signatures in tumor DNA. In the second aim, we will compare the types of CRs formed in response to a stalled replication fork with CRs resulting from endonuclease-induced DSBs. We will use the bacterial Tus/Ter system to create a site-specific stalled replication fork in yeast or human cells, identify the resulting types of CRs and the factors that suppress different CR outcomes. The powerful genetic screens proposed should enable identification of mutational events (base substitutions and indels) in addition to CRs and we will also characterize these events and the repair pathways that suppress their formation. We expect the knowledge garnered from these studies will aid in identifying new mutational signatures and their underlying pathologies.

总结 大多数人类癌细胞表现出基因组不稳定性，从突变率升高（碱基替换 和插入缺失）与染色体重排（CR）和非整倍性的关系。下一代的应用 测序（NGS）技术来分析癌症基因组已经产生了大量的信息， 谱的基因组不稳定性，并导致识别特定的突变和CR签名相关 不同DNA修复途径的缺失。普遍的观点是，CR是通过容易出错的 处理受损的染色体。虽然CR的起始病变的性质尚不清楚，但许多 来自酵母和人类细胞的遗传证据表明DNA复制错误是病毒的来源 断裂的染色体为CR提供能量本提案中概述的两个目标将解决启动的根源问题， CRs的损伤，并测量CRs在野生型和修复缺陷细胞中的频率和光谱， 对DNA损伤的反应。在第一个目标中，我们将使用CRISPR-Cas9诱导位点特异性DSB。 酵母或人类细胞，并确定存活细胞中CR的全谱。实验将是 在缺乏非同源末端连接（NHEJ）的特定组分的细胞中进行，同源依赖性 修复（HR）或DNA损伤信号通路，以确定不同类型的CR如何被抑制。我们 期望为每种缺陷定义唯一的CR签名，以指导识别新型CR 肿瘤DNA中的特征在第二个目标，我们将比较类型的CR形成，以应对一个停滞的 具有由内切核酸酶诱导的DSB产生的CR的复制叉。我们将使用细菌Tus/Ter系统 为了在酵母或人类细胞中产生位点特异性停滞复制叉，鉴定所产生的CR类型， 抑制不同CR结局的因素。所提出的强大的基因筛选应该能够 除了CR之外，我们还将鉴定突变事件（碱基置换和插入缺失）， 表征这些事件和抑制其形成的修复途径。我们期待知识 从这些研究中获得的信息将有助于确定新的突变特征及其潜在的病理学。