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，年龄发作的活性氧增加导致DNA上的遗传毒性应激增加，这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，无法修复这些DNA损伤加合物，因此损伤的年龄发作性积累是疾病的触发因素，与由于聚ADP核糖聚合反应和烟酰胺腺嘌呤二核苷酸（NAD+）水平的排出引起的代谢能量应激有关。 利用我们过去在活细胞中使用荧光寿命成像测量（FLIM）的生物光子方法来量化荧光标记蛋白质的Forster共振能量转移（FRET）的成功，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损伤响应和分辨发生在秒的时间间隔内，在DNA损伤的近端空间小于10 nm，因此完全在FLIM-FRET测量的空间和时间参数内。我们寻求开发新的技术来直接观察蛋白质在100- 10 nm空间的空间位置。 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。该目标的目的是以尽可能接近内源化学计量的方式在活的人类细胞中标记蛋白质。这是可能的，通过避免荧光蛋白融合更明亮的荧光染料标记的蛋白质。在DNA上，我们将以亚化学计量的染料量标记DNA，或者使用组蛋白H2 B-荧光蛋白融合物标记染色质，测试潜在的FRET对以确定哪些对是最佳的。我们将比较这个信号与荧光蛋白融合。这是一种通用的技术，适用于任何DNA损伤蛋白。 第二个目标是开发一种实时观察活细胞中DNA损伤反应的方法。我们将使用的系统是人HTRT永生化细胞。我们将使用一系列特定的DNA损伤剂，通过定位于DNA来确定哪种类型的损伤与测试的每种疾病蛋白最相关。对于重组蛋白，我们将使用由合作者提供或内部生产的CryoEM质量纯蛋白。随着时间的推移，这种定位将被捕获并与结构照明显微镜（SIM）的超分辨率成像相关。这种方法将适用于任何DNA修复因子。 最终目标是对DNA修复和细胞代谢进行真实的成像。我们将以每秒一帧或更少的速度进行时间观察，空间观察在100 A以下，以成像疾病蛋白质募集到DNA损伤的动力学，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水平将通过损伤和修复过程中的NAD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.