Project 2 Symington

摘要 大多数人类癌细胞表现出基因组的不稳定性，从高的突变率(碱基替换 与染色体重排(CRS)和非整倍体有关。浅谈下一代网络的应用 用于分析癌症基因组的测序(NGS)技术已经产生了关于 基因组不稳定性的光谱，并导致特定突变和相关CR签名的鉴定 伴随着不同DNA修复途径的丢失。主流观点认为，CRS是通过容易出错的方式生成的 处理受损的染色体。尽管CRS的起始损害的性质尚不清楚，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 支持CRS的染色体断裂。本提案中概述的两个目标将解决启动的来源 损伤CRS，并测量CRS在野生型和修复缺陷细胞中的频率和频谱 对明确的DNA损伤的反应。在第一个目标中，我们将使用CRISPR-Cas9在 酵母或人类细胞，并测定存活细胞中CRS的全谱。这些实验将是 在缺乏非同源末端连接(NHEJ)特定成分的细胞中进行，同源依赖 修复(HR)或DNA损伤信号通路，以确定不同类型的CRS是如何被抑制的。我们 希望为每个缺陷定义唯一的CR签名，以指导新CR的识别 肿瘤DNA中的特征。在第二个目标中，我们将比较因应对停滞而形成的CRS的类型 由核酸内切酶诱导的DSB引起的CRS的复制叉子。我们将使用细菌Tus/Ter系统 为了在酵母或人类细胞中创建特定部位的停滞复制叉子，确定产生的CRS类型和 抑制不同CR结局的因素。提出的强大的基因筛查应该能够 除了CRS，我们还将鉴定突变事件(碱基替换和INDELs) 描述这些事件以及抑制其形成的修复途径。我们希望了解到 从这些研究中获得的信息将有助于识别新的突变特征及其潜在的病理机制。

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.