Q-FADD: A Mechanistic Approach for Modeling the Accumulation of Proteins at Sites of DNA Damage.
Q-FADD: A Mechanistic Approach for Modeling the Accumulation of Proteins at Sites of DNA Damage.
复制标题
Q-FADD：一种模拟 DNA 损伤位点蛋白质积累的机制方法。
DOI：
10.1016/j.bpj.2019.04.032
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发表时间：
2019
影响因子：
3.4
通讯作者：
Luger,Karolin
中科院分区：
文献类型：
--
作者：
Mahadevan,Jyothi;Rudolph,Johannes;Jha,Asmita;Tay,JianWei;Dragavon,Joseph;Grumstrup,ErikM;Luger,Karolin
DNA损伤的修复需要许多不同蛋白质的有序募集，这些蛋白质负责信号传导和随后的修复。一个强大的和广泛使用的工具，用于研究这些蛋白质在损伤部位的协调积累是活细胞中的激光显微照射，然后监测荧光标记的蛋白质的积累问题。尽管这一方法得到广泛使用，但没有严格的方法来定量描述征聘过程。在这里，我们介绍了一个扩散模型，明确说明了独特的大小和形状的单个细胞核，并使用两个变量：Deff，有效的扩散系数，和F，移动的蛋白质的分数，积累在DNA损伤的网站。我们的模型定量描述了三种测试蛋白质：聚ADP-核糖聚合酶1和2（PARP 1/2）和组蛋白PARylation因子1的积累。通过我们的方法推导出的PARP 1的Deff比PARP 2大6倍，并且与使用荧光相关光谱和光漂白后荧光恢复的先前文献报道一致。我们的数据表明，组蛋白PAR化因子1到达DNA损伤的网站独立于任何PARP。重要的是，我们的模型可以应用于现有数据，可以直接比较不同细胞类型之间的任何DNA修复蛋白的扩散系数，在不同的实验室和不同的方法中获得，并且还可以询问细胞间的变异性。
The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful and widely used tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing the recruitment process quantitatively. Here, we introduce a diffusion model that explicitly accounts for the unique sizes and shapes of individual nuclei and uses two variables: Deff, the effective coefficient of diffusion, and F, the fraction of mobile protein that accumulates at sites of DNA damage. Our model quantitatively describes the accumulation of three test proteins, poly-ADP-ribose polymerases 1 and 2 (PARP1/2) and histone PARylation factor 1. Defffor PARP1, as derived by our approach, is 6× greater than for PARP2 and in agreement with previous literature reports using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Our data indicate that histone PARylation factor 1 arrives at sites of DNA damage independently of either PARP. Importantly, our model, which can be applied to existing data, allows for the direct comparison of the coefficient of diffusion for any DNA repair protein between different cell types, obtained in different laboratories and by different methods, and also allows for the interrogation of cell-to-cell variability.