Decoding Genome Instability by Combining Accurate Mapping and Predictive Modeling

摘要 DNA双链断裂(DSB)是DNA损伤最致命的形式，会导致衰老和癌症。A Main 自发性DSB的来源是复制应激，即DNA复制中的异常导致速度减慢或停滞 复制分叉。复制应激可以直接或间接地引起DSB，细胞对它的反应是 通常是异构性的，这会导致难以解释的DSB模式。为了克服这一挑战，我们 将使用计算机模拟来分析DSB数据并推断DSB产生的潜在机制。我们会 建立和扩展我们在上一个资助期开发的技术：(1)i-blost：最敏感的 DSB检测方法，允许在100,000个细胞中检测到1个DSB；(2)定量DSB测序：唯一 允许在全基因组范围内精确测量绝对DSB频率(DSB/细胞)的方法；以及(3) Repli-Sim：对DNA复制的大规模计算机模拟，准确地复制单细胞和 全人口的数据。具体地说，我们将使用创新的计算方法和 1)阐明了DNA复制的时空调控机制 以及它的干扰如何导致复制压力。2)澄清和量化复制压力的后果和 对产生的DSB进行分类3)表征由以下原因引起的DSB的细胞群体分布的异质性 复制应激并推断其潜在机制。我们的大规模研究将使我们能够将每个 将个人成果纳入所取得的所有其他成果的更广泛的背景中，从而加深了对其的解释和 从而允许对所获得的DSB景观和调节复制的推断路径进行分类。已被占用 总之，我们的结果将导致对导致和预防的机制的系统层面的理解 复制应激和DSB。我们的项目将通过量化将基因组不稳定性的研究提高到一个新的水平 复制应激的机制及其如何导致DSB。我们的工作还将提供方法和 进一步研究基因组不稳定性的计算工具，并为使用这些知识指导 治疗决定。

ABSTRACT DNA double-strand breaks (DSBs) are the most lethal form of DNA damage and drive aging and cancer. A main source of spontaneous DSBs is replication stress, i.e. aberrations in DNA replication leading to slowing or stalling of replication forks. Replication stress can cause DSBs both directly and indirectly, and cells’ reaction to it is often heterogeneous, which leads to DSB patterns that are difficult to interpret. To overcome this challenge, we will use computer simulations to analyze DSB data and infer underlying mechanisms of DSB creation. We will build on and expand techniques we developed in the previous funding period: (1) i-BLESS: the most sensitive DSB detection method, allowing detection of 1 DSB in 100,000 cells; (2) quantitative DSB sequencing: the only approach that allows precise genome-wide measurement of absolute DSB frequencies (DSBs/cell); and (3) Repli-Sim: massive computer simulations of DNA replication that accurately reproduce both single-cell and population-wide data. Specifically, we will use a combination of innovative computational methods and experiments in the following Aims: 1) Elucidate the mechanisms of spatiotemporal regulation of DNA replication and how its disturbance causes replication stress. 2) Clarify and quantify consequences of replication stress and classify resulting DSBs 3) Characterize heterogeneity of cell population distribution of DSBs resulting from replication stress and infer its underlying mechanisms. The large-scale of our study will allow us to put each individual result into much broader context of all other results obtained, thus deepening its interpretation and allowing for classification of obtained DSB landscapes and inferred pathways regulating replication. Taken together, our results will lead to a system-level understanding of the mechanisms that cause and prevent replication stress and DSBs. Our project will raise the study of genomic instability to a new level by quantifying the mechanisms of replication stress and how they lead to DSBs. Our work will also provide methods and computational tools to further study genome instability and pave the way to use this knowledge to guide therapeutic decisions.