A Dynamic Perspective of the DNA Damage Response

摘要 DNA 损伤修复机制是高度动态的，有许多相互作用的成分。然而，它 目前尚不清楚各种修复模块如何动态交互。对这些有了更深入的了解 机制可以让我们最大限度地利用这些动态来获得治疗益处。我开发了一个 创新的高通量筛选技术，全基因组动态评估（GEoDE），结合 高密度 6,144 菌落酵母菌落遗传突变体阵列，具有延时成像功能，可实现基因组- 广泛的、时间分辨的健康筛选，可用于筛选多种治疗药物 实时显示数千个殖民地。使用该技术分析紫外线诱导的 DNA 损伤反应 阐明了酵母中 UV 响应途径的动态测序并鉴定了 175 种新型 UV 响应 基因。重要的是，GEoDE 可以识别每个基因从 UV 恢复所需的时间。 诱发的损害。这些发现表明遗传适应性景观是动态的并且包含有价值的 时间和条件特定信息。我们实验室构建的另一个工具，系统基因到表型阵列 （SGPA），允许询问给定表型背后的遗传景观。该提案使用 GEoDE 和 SGPA 将首先通过全基因组筛选建立 DNA 损伤动态响应的完整模型 遗传上易于处理的模式生物酿酒酵母（目标 1），然后通过人体细胞中的靶向筛选 线（目标 2）。最终目标是确定 DNA 中的特定治疗靶点和治疗窗口 损伤反应途径。

ABSTRACT The DNA damage repair mechanism is highly dynamic, with many interacting components. However, it remains unclear how various repair modules dynamically interact. A greater understanding of these mechanisms could allow us to maximally leverage these dynamics for therapeutic benefit. I have developed an innovative high-throughput screening technique, Genome-Wide Dynamic Evaluation (GEoDE), that couples high-density 6,144 colony yeast colony genetic mutant arrays with time-lapse imaging to achieve genome- wide, time-resolved fitness screens which may be used to screen multiple therapeutic agents against thousands of colonies in real time. Analysis of the UV-induced DNA damage response using this technique has elucidated the dynamic sequencing of UV response pathways in yeast and identified 175 novel UV-responding genes. Importantly, GEoDE can identify the time at which each gene is needed in the recovery from UV- induced damage. These findings suggest that genetic fitness landscapes are dynamic and contain valuable time and condition-specific information. An additional tool built by our lab, systematic-gene-to-phenotype arrays (SGPA), permits interrogation of genetic landscapes behind given phenotype. This proposal uses GEoDE and SGPA to build a complete model of the dynamic response to DNA damage, first via genome-wide screens in the genetically tractable model organism S. cerevisiae (Aim 1), and then via targeted screens in human cell lines (Aim 2). The ultimate goal is to identify specific therapeutic targets and treatment windows in the DNA damage response pathway.