Scanning Correlation Microscopy Methods for Quantifying DNA Repair Kinetics

描述（由申请人提供）：大多数荧光标记的DNA修复蛋白暴露于电离辐射或拟辐射化学剂后的动力学，如果不严重干扰所研究的系统，就不能在活细胞中量化。除了少数蛋白在DNA损伤位点附近大量附着（如3-H2AX， 53BP1）外，大多数其他蛋白在DNA损伤位点附近附着的拷贝数较少，无法通过荧光显微镜观察到。这是因为自由移动或不移动的荧光蛋白的高背景掩盖了DNA修复蛋白在损伤部位的弱聚集。因此，DNA损伤通常是通过激光照射诱导聚集性DNA损伤来可视化的。这种光照诱导修复蛋白在细胞核的一个或多个大的斑点上大量积累。然而，激光引起的损伤在本质上是复杂的，并不能很好地代替电离辐射或模拟辐射来研究体内DNA修复。在这项工作中，我们建议开发定量显微镜方法，以克服目前无法量化稀疏DNA损伤位点荧光标记DNA修复蛋白动力学的主要限制。更具体地说，我们将应用光栅图像相关光谱（RICS），一种分析图像像素的时空荧光强度波动的技术，来量化细胞核中稀疏积累的蛋白质的修复动力学。我们将使用RICS来量化dna依赖性蛋白激酶催化亚基（DNA-PKCS）在暴露于3射线和博来霉素（均为双链断裂形成剂）后的野生型和修复缺陷7A形式的修复动力学。我们还将测试双氧水，单链断裂（DSB）形成剂，作为阴性对照。我们将证明DSBs形成后的DNA-PKCS动力学可以通过RICS量化。此外，我们建议开发两种特殊形式的RICS，以进一步加强这些蛋白质的DNA修复动力学的量化：(i)光激活RICS （PA-RICS）将提供对荧光标记修复蛋白浓度的控制；（ii）相干控制RICS （CC-RICS）将优化激光脉冲特性，在不增加激发功率的情况下提高荧光发射的数量级。PA-RICS和CC-RICS将改进DNA- pkcs变体在DNA损伤位点的结合动力学常数的量化。重要的是，所提出的RICS技术可以潜在地用于量化各种DNA损伤传感、信号传导和修复蛋白的动力学，这些蛋白在细胞核中具有稀疏的积累模式。因此，所提出的方法非常普遍适用于DNA修复领域及其他领域。

DESCRIPTION (provided by applicant): The kinetics of most fluorescently tagged DNA repair proteins subsequent to exposure to ionizing radiation, or radiomimetic chemical agents, cannot be quantified in the living cell without serious perturbation of the system under study. With the exception of only few proteins that attach in large numbers near DNA damage sites (e.g. 3-H2AX, 53BP1), most other proteins attach in fewer copies near the DNA damage sites and cannot be visualized by fluorescence microscopy. This because of the high background from freely moving, or immobile, fluorescent proteins that mask the weak aggregation of DNA repair proteins at damage sites. As a result, DNA damage is usually visualized by inducing clustered DNA damage by illumination with a laser beam. This illumination induces very high accumulation of repair proteins in one or more large spots in the nucleus. Nevertheless, the laser-induced damage is complicated in nature and may not be a good surrogate to ionizing radiation or radiomimetic agents for studying DNA repair in vivo. In this work we propose to develop quantitative microscopy methods that can overcome the current major limitation of not being able to quantify the kinetics of fluorescently tagged DNA repair proteins at sparse DNA damage sites. More specifically we will apply raster image correlation spectroscopy (RICS), a technique that analyzes the spatio-temporal fluorescence intensity fluctuations in image pixels, to quantify the repair kinetics of proteins with sparse accumulation in the cell nucleus. We will use RICS to quantify the repair kinetics of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS), in its wild type and repair-deficient 7A forms after exposure to 3-rays and bleomycin that are both double strand break forming agents. We will also test hydrogen peroxide, a single strand break (DSB) forming agent, as a negative control. We will show that the DNA-PKCS kinetics after formation of DSBs can be quantified by RICS. Furthermore we propose to develop two specialized forms of RICS to further enhance the quantification of DNA repair kinetics of these proteins: (i) Photo-Activation RICS (PA-RICS) will offer control of the fluorescently tagged repair protein concentration, and (ii) Coherent Control RICS (CC-RICS) will optimize laser pulse characteristics to enhance fluorescence emission by up to an order of magnitude without increasing excitation power. PA-RICS and CC-RICS will enable improved quantification of the binding kinetic constants of DNA-PKCS variants at DNA damage sites. Importantly, the proposed RICS techniques can potentially be used to quantify the kinetics of a wide range of DNA damage sensing, signaling, and repair proteins with sparse accumulation patterns in the nucleus. Therefore, the proposed methods are very generally applicable to the DNA repair field and beyond.