Automated modeling of protein accumulation at DNA damage sites using qFADD.py.
Automated modeling of protein accumulation at DNA damage sites using qFADD.py.
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DOI：
10.1017/s2633903x22000083
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发表时间：
2022-01-01
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影响因子：
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通讯作者：
Luger, Karolin
中科院分区：
文献类型：
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作者：
Bowerman, Samuel;Mahadevan, Jyothi;Luger, Karolin
真核细胞经常受到DNA损伤，常常对生物体的健康造成有害的后果。细胞通过多种修复途径来减轻这种DNA损伤，这些修复途径涉及多种和大量不同的蛋白质。为了更好地理解细胞对DNA损伤的反应，人们需要精确测量这些修复蛋白的积累、保留和耗散时间尺度。在这里，我们描述了一种自动实现的“DNA损伤后荧光积累的定量”方法，该方法大大增强了广泛使用的激光微照射技术的分析和定量，该技术用于研究DNA修复蛋白在DNA损伤位点的招募。这个开源实现（“qFADD.py”）是一个独立的软件包，可以在笔记本电脑或计算机集群上运行。我们的实现包括对核漂移的修正，对最佳拟合模型的自动网格搜索，以及对水平条纹和斑点实验进行建模的能力。为了提高统计的严谨性，网格搜索算法还包括对重复的自动模拟。作为一个实际的例子，我们提出并讨论了早期应答者PARP1对DNA损伤位点的招募动态。
Eukaryotic cells are constantly subject to DNA damage, often with detrimental consequences for the health of the organism. Cells mitigate this DNA damage through a variety of repair pathways involving a diverse and large number of different proteins. To better understand the cellular response to DNA damage, one needs accurate measurements of the accumulation, retention, and dissipation timescales of these repair proteins. Here, we describe an automated implementation of the "quantitation of fluorescence accumulation after DNA damage" method that greatly enhances the analysis and quantitation of the widely used technique known as laser microirradiation, which is used to study the recruitment of DNA repair proteins to sites of DNA damage. This open-source implementation ("qFADD.py") is available as a stand-alone software package that can be run on laptops or computer clusters. Our implementation includes corrections for nuclear drift, an automated grid search for the model of a best fit, and the ability to model both horizontal striping and speckle experiments. To improve statistical rigor, the grid-search algorithm also includes automated simulation of replicates. As a practical example, we present and discuss the recruitment dynamics of the early responder PARP1 to DNA damage sites.