A high throughput assay to identify novel chromatin factors that regulate DNA repair

对我们DNA的破坏是对我们细胞功能的最直接威胁之一 正常情况下，以及我们将遗传信息忠实地传递给后代的能力。我们的细胞 经历大量的自发性DNA损伤。正如我们所知，生活是可能的，谢谢 真核细胞进化出的特定DNA修复机制。这种机制涉及到 与信号协同工作的传感器、放大器和效应器的高度协调动作 允许有效DNA修复的途径，即所谓的DNA损伤反应(DDR)。我们的 DNA密集地堆积在染色质中，这是一种需要克服的障碍，需要正确识别和 DNA损伤的修复。事实上，染色质动力学已经成为 DDR，然而，直接参与修复过程的特定染色质因子仍然 鲜为人知。在这方面，这一领域的进展一直受到可获得的有限的 高通量技术。在这项探索性拨款中，我们将尝试通过以下方式解决这些限制 建立染色质因子库和基于显微镜的高通量分析 遵循染色质因子对DNA断裂的动力学和募集。具体地说，在目标1中，我们将 开发染色质因子库(ChromORFeome)并使用高通量激光破碎机 鉴定被招募到DNA断裂部位的新的染色质因子。目标2将提供以下证据 这些新鉴定的染色质因子的原理验证。这笔探索性拨款的结果将 建立先进的新技术来研究DNA修复过程并鉴定新的 染色质因子在这一过程中起着关键作用。

Damage to our DNA poses one of the most direct threats to both the ability of our cells to function normally, as well as our ability to faithfully transmit genetic information to our progeny. Our cells experience a significant amount of spontaneous DNA lesions. Life, as we know it, is possible thanks to specific DNA repair mechanisms that eukaryotic cells have evolved. Such mechanisms involved the highly coordinated action of sensors, amplifiers and effectors that work in concert with signaling pathways to allow efficient DNA repair, in what is known as the DNA Damage Response (DDR). Our DNA is densely packed in chromatin, a barrier that needs to be overcome for proper recognition and repair of DNA lesions. Indeed, chromatin dynamics has emerged as an important module in the DDR, however the specific chromatin factors that are directly involved in the repair process remain poorly known. In this context, advances in the field have been hampered by limited availability of high-throughput technologies. In this exploratory grant, we will attempt to tackle these limitations by developing both a library of chromatin factors and a microscope based-high throughput assay to follow kinetics and recruitment of chromatin factors to DNA breaks. Specifically, in Aim 1 we will develop a library (ChromORFeome) of chromatin factors and use a high-throughput laser breaks assay to identify novel chromatin factors recruited to sites of DNA breaks. Aim 2 will provide proof of principle validation of these novel identified chromatin factors. Results from this exploratory grant will establish advanced new technologies to study the process of DNA repair and identify novel chromatin factors that play key roles in this process.