Development of a Novel Assay to Measure DSB Repair at Endogenous Loci in Cells

 描述(申请人提供)：本R03申请的目标是开发一种新的方法来测量内源性基因座的DSB修复，使用我们实验室最近开发的下一代测序平台和自动比对程序。这种方法与以前描述的分析方法不同，因为它不需要将人工序列整合到基因组中，也不依赖于荧光蛋白作为报告。因此，我们的检测方法可以迅速应用于许多不同的细胞系，甚至未来的原代肿瘤培养。因此，我们的方法将同时用于基础科学和临床应用。双链断裂是对基因组完整性和细胞生存的严重威胁，因此已经进化出复杂的通路，可以快速检测DNA断裂，激活细胞周期检查点来阻止生长，然后修复这些损伤。此外，肿瘤细胞的存活率往往 依赖于抗癌治疗，如电离辐射(IR)和许多化疗药物引起的DSB的修复。因此，人们对阐明参与DSB修复的关键蛋白以及它们的作用机制一直有着浓厚的兴趣。这些见解对于更好地理解哺乳动物细胞中的DNA损伤反应以及开发新的癌症疗法将是重要的。哺乳动物细胞中有两条关键的DSB修复途径，即非同源末端连接(NHEJ)和同源重组(HR)。对这些途径中的关键分子有很多见解，甚至最近发现许多新的蛋白质在DSB的识别、分类和修复中发挥重要作用。DSB修复分析对这些研究工作至关重要，需要更好的分析来报告发生在基因组内源性位置的修复事件。我于2012年8月开始我的实验室，在我们的头两年里，我们开发了一种新的方法来评估基因组座位上的DSB修复，使用下一代测序和自动序列比对。在初步研究中，我们创建了下一代测序方案，并证明了在细胞中设计核酸酶位点的初步可行性。在目标1中，我们将调整我们的平台，以在多个内源基因组位置测量NHEJ。在目标2中，我们将进一步设计我们的系统，在基因组中自然出现的侧翼序列同源性区域测量NHEJ和HR。我们建议开发一种全新的方法来测量DSB修复，它不需要将序列整合到基因组中，也不依赖于荧光蛋白作为报告。不依赖于荧光蛋白作为读数，这意味着DSB修复活性可以几乎即时地、在多个时间点以良好的分辨率进行评估。R03中提出的实验将作为一个关键的原则证明，使用基于下一代测序的方法，可以在内源基因座测量突变的NHEJ和HR。因此，它们与支持开发新的研究技术和方法的R03拨款机制是一致的。

 DESCRIPTION (provided by applicant): The goal of this R03 application is to develop a novel method to measure DSB repair at endogenous loci, using a next-generation sequencing platform and automated alignment program that was recently developed in our laboratory. This method is unique from previously described assays, because it does not require the integration of artificial sequences into the genome, and it does not rely on a fluorescent protein as a reporter. As such, our assay could be rapidly applied to many different cell lines, and even primary tumor cultures in the future. Thus, our approach will have uses for both basic science and clinical applications. DSBs are a serious threat to genomic integrity and cell survival, and thus complex pathways have evolved which can rapidly detect DNA breaks, activate cell cycle checkpoints to arrest growth, and then repair these lesions. In addition, tumor cell survival often is dependent on the repair of DSBs induced by anti-cancer treatments, such as ionizing radiation (IR) and many chemotherapeutics. As such, there has been intense interest in elucidating the key proteins involved in DSB repair, and the mechanisms by which they function. Such insights would be important for a better understanding of the DNA damage response in mammalian cells and in developing novel cancer therapeutics. There are two key DSB repair pathways in mammalian cells, non- homologous end joining (NHEJ) and homologous recombination (HR). There have been numerous insights into the key players in these pathways, and even recently many new proteins were found to play important roles in the recognition, triage and repair of DSBs. DSB repair assays are critical for these research efforts, and better assays are needed which report on repair events that occur at endogenous sites in the genome. I started my laboratory in August of 2012, and during our first 2 years we developed a novel approach to assess DSB repair at genomic loci using next-generation sequencing and automated sequence alignment. In preliminary studies, we created a next-generation sequencing protocol and we demonstrated initial feasibility with an engineered nuclease site in cells. In Aim 1, we will adapt our platform to measure NHEJ at multiple endogenous genomic sites. In Aim 2, we will further engineer our system to measure both NHEJ and HR at a naturally occurring region of flanking sequence homology in the genome. We are proposing to develop a completely novel method to measure DSB repair, which does not require the integration of sequences into the genome, and which does not rely on a fluorescent protein as a reporter. The lack of dependence on a fluorescent protein as a read-out means that DSB repair activity can be assessed nearly instantaneously, and at multiple time-points with fine resolution. The experiments proposed in this R03 will serve as a critical proof-of-principle that both mutagenic NHEJ and HR can be measured at endogenous loci using a next-generation sequencing-based approach. Thus, they are consistent with the R03 grant mechanism which supports the development of new research technology and methodology.