ABSTRACT Fluorescence microscopy in combination with the induction of localized DNA damage using focused light beams has played a major role in the study of protein recruitment kinetics to DNA damage sites in recent years. Currently published methods are dedicated to the study of single fluorophore/single protein kinetics. However, these methods may be limited when studying the relative recruitment dynamics between two or more proteins due to cell-to-cell variability in gene expression and recruitment kinetics, and are not suitable for comparative analysis of fast-recruiting proteins. To tackle these limitations, we have established a time-lapse fluorescence microscopy method based on simultaneous dual-channel acquisition following UV-A-induced local DNA damage coupled with a standardized image and recruitment analysis workflow. Simultaneous acquisition is achieved by spectrally splitting the emitted light into two light paths, which are simultaneously imaged on two halves of the same camera chip. To validate this method, we studied the recruitment of poly(ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) glycohydrolase (PARG), proliferating cell nuclear antigen (PCNA) and the chromatin remodeler ALC1. In accordance with the published data based on single fluorophore imaging, simultaneous dual-channel imaging revealed that PARP1 regulates fast recruitment and dissociation of PARG and that in PARP1-depleted cells PARG and PCNA are recruited with comparable kinetics. This approach is particularly advantageous for analyzing the recruitment sequence of fast-recruiting proteins such as PARP1 and ALC1, and revealed that PARP1 is recruited faster than ALC1. Split-view imaging can be incorporated into any laser microirradiation-adapted microscopy setup together with a recruitment-dedicated image analysis package.

中文翻译：

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同时双通道成像量化激光诱导 DNA 损伤位点的相互依赖的蛋白质募集

摘要 近年来，荧光显微镜结合使用聚焦光束诱导局部 DNA 损伤在 DNA 损伤位点的蛋白质募集动力学研究中发挥了重要作用。目前发表的方法致力于研究单荧光团/单蛋白质动力学。然而，由于基因表达和募集动力学的细胞间变异性，这些方法在研究两种或多种蛋白质之间的相对募集动态时可能会受到限制，并且不适合快速募集蛋白质的比较分析。为了解决这些限制，我们建立了一种延时荧光显微镜方法，该方法基于 UV-A 诱导的局部 DNA 损伤后的同步双通道采集，并结合标准化的图像和招募分析工作流程。通过将发射光光谱分成两条光路来实现同时采集，这两条光路在同一相机芯片的两半上同时成像。为了验证这种方法，我们研究了聚（ADP-核糖）聚合酶 1（PARP1）、聚（ADP-核糖）糖水解酶 (PARG)、增殖细胞核抗原 (PCNA) 和染色质重塑剂 ALC1 的募集。根据基于单荧光团成像的已发表数据，同步双通道成像显示 PARP1 调节 PARG 的快速募集和解离，并且在 PARP1 耗尽的细胞中，PARG 和 PCNA 的募集具有可比的动力学。这种方法特别有利于分析 PARP1 和 ALC1 等快速募集蛋白的募集序列，并表明 PARP1 的募集速度比 ALC1 更快。