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

点击翻译按钮获取中文摘要

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.