A Dynamic Perspective of the DNA Damage Response

摘要 DNA损伤修复机制是高度动态的，具有许多相互作用的组分。但 目前还不清楚各种修复模块如何动态地相互作用。更好地理解这些 机制可以让我们最大限度地利用这些动力学来获得治疗益处。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）， 高密度6，144菌落酵母菌落遗传突变体阵列与延时成像，以实现基因组- 广泛的，时间分辨的健身筛选，可用于筛选多种治疗剂， 在真实的时间里产生成千上万的殖民地。使用该技术分析UV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种新的UV响应途径。 基因.重要的是，GEoDE可以确定每个基因在从紫外线中恢复所需的时间。 诱发的损害。这些发现表明，遗传适应度景观是动态的， 时间和条件特定的信息。我们实验室建立的另一个工具，系统基因-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， SGPA建立了一个完整的模型，动态响应DNA损伤，首先通过基因组范围的屏幕， 遗传上易处理的模式生物S.酿酒酵母（Aim 1），然后通过在人细胞中的靶向筛选， 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.