Development of live cell FLIM-FRET to Characterize Protein-DNA Interactions During DNA Damage Repair

我们的假设是，在人类中，年龄起病的活性氧增加导致并增加了对DNA的遗传毒性应激，这种应激必须通过核苷酸切除修复途径的碱基切除修复来修复。在氧化应激期间，不能修复这些DNA损伤加合物，从而导致损伤的年龄积累，是疾病的触发因素，与多聚ADP核糖聚合反应引起的代谢能量应激和烟酰胺腺嘌呤二核苷酸(NAD+)水平的排出有关。利用我们过去在活细胞中利用荧光寿命成像测量(FLIM)技术定量福斯特共振能量转移(FRET)的成功经验，我们寻求开发与DNA损伤修复相关的蛋白质-蛋白质和蛋白质-DNA相互作用的实时视频速度观察。DNA损伤的响应和分解发生在数秒的时间空间内，发生在DNA损伤的10 nm以下的近端空间，因此完全在Flim-FRET测量的空间和时间参数范围内。我们寻求开发新的技术，在100-10 nm的空间直接观察蛋白质的空间位置。第一个目标是将重组的纯蛋白质直接荧光标记到活细胞中。这一目标的目的是在活的人类细胞中以尽可能接近内源性化学计量的方式转导标记的蛋白质。这可以通过避免荧光蛋白与更明亮的荧光染料标记的蛋白融合来实现。在DNA中，我们将以亚化学计量比的染料标记DNA，或使用组蛋白H2B-荧光蛋白融合来标记染色质，测试潜在的FRET对以确定哪些对是最佳的。我们将把这个信号与荧光蛋白融合进行比较。这是任何DNA损伤蛋白的通用技术。第二个目标是开发一种在活细胞中实时观察DNA损伤反应的方法。我们将使用的系统是人类hTERT永生化细胞。我们将使用一系列特定的DNA损伤剂，通过对DNA的定位来确定哪种类型的损伤与所测试的每种疾病蛋白最相关。对于重组蛋白质，我们将使用合作伙伴提供的或内部生产的冷冻EM质量的纯蛋白质。随着时间的推移，这种定位将被捕捉并与结构照明显微镜(SIM)的超分辨率成像相关联。这种方法将适用于任何DNA修复因子。最终目标是实时成像DNA修复和细胞新陈代谢。我们将以每秒一帧或更少的速度进行时间观测，并在100A以下进行空间观测，以成像疾病蛋白质募集到DNA损伤的动力学，以及重要的是，染色质的分辨率。在此背景下，我们将测试各种DNA损伤调节剂。NAD水平将通过NAD在损坏和修复过程中的荧光寿命来测量。

Our hypothesis is that in humans, age-onset reactive oxygen species increase is leading to and increased genotoxic stress on DNA, that must be repaired by either/or base excision repair of nucleotide excision repair pathways. During oxidative stress the inability to repair these DNA damage adducts, hence age-onset accumulation of damage, is the trigger of disease, linked to metabolic energy stresses due the poly-ADP ribose polymerization response and draining of nicotinamide adenine dinucleotide (NAD+) levels. Using our past success with biophotonic methods in live cells with Fluorescence Lifetime Imaging Measurement (FLIM) to quantify Forster Resonant Energy Transfer (FRET) with fluorescently labeled proteins, we seek to develop a real-time video speed observation of protein-protein and protein-DNA interactions relevant to DNA damage repair. DNA damage response and resolution take place in a temporal space of seconds, in proximal space of under 10nm to DNA damage, thus well within the space and time parameters of FLIM-FRET measure. We seek to develop new technology to directly observed protein spatial locations in the space of 100-10nm. The first aim is to transduce recombinant, pure proteins, directly fluorescently labelled into live cells. The goal of this aim is to transduce labelled proteins in live human cells at closely as possible to endogenous stoichiometry. This is possible by avoiding fluorescent protein fusions with much brighter fluorescent dye-labeled protein. At the DNA, we will either label DNA at sub-stoichiometric amounts of dye or use histone H2B-fluorescent protein fusions to label chromatin, testing potential FRET pairs to determine which pairs are optimal. We will compare this signal to fluorescent protein fusions. This is a universal technique for any DNA damage protein. The second aim is to develop an assay observe real-time DNA damage response in live cells. The system we will use are human HTERT immortalized cells. We will use a series of specific DNA damage agents to determine by localization to DNA which type of damage is most relevant for each disease protein tested. For recombinant protein, we will be using CryoEM quality pure proteins provided by collaborators or produced in-house. This localization over time will be captured and correlated to super-resolution imaging by structured illumination microscopy (SIM). This methodology will be applicable to any DNA repair factor. The final aim is to image DNA repair and cell metabolism in real time. We will make temporal observations at one frame per second, or less, with spatial observations under 100A to image the kinetics of disease protein recruitment to DNA damage and importantly, the resolution off chromatin. Within this context, we will test various DNA damage modulators. NAD levels will be measured by NAD fluorescent lifetime over the course of damage and repair.