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是如何动态相互作用的。更好地理解这些 这些机制可以让我们最大限度地利用这些动态来获得治疗效益。我已经开发出一种 创新的高通量筛选技术，基因组范围动态评估(GEODE)，将 高密度6,144菌落酵母菌落遗传突变阵列与延时成像实现基因组- 宽大的、时间分辨率的健身屏幕，可用于筛选多种治疗药物 数以千计的殖民地实时运行。使用这项技术分析紫外线诱导的DNA损伤反应 阐明酵母中紫外光响应途径的动态测序，并鉴定出175个新的紫外光响应信号 基因。重要的是，Geode可以识别每个基因在从紫外线中恢复所需的时间。 诱导性损害。这些发现表明，遗传适应性是动态的，包含有价值的 特定于时间和条件的信息。我们实验室开发的另一种工具，系统化基因-表型阵列 (SGPA)，允许询问给定表型背后的遗传景观。该方案使用geode和 SGPA将首先通过全基因组筛选建立对DNA损伤的动态反应的完整模型 遗传上易处理的模式生物酿酒酵母(Aim 1)，然后通过在人类细胞中的靶向筛选 线路(目标2)。最终目标是在DNA中确定特定的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.