Assessment of DNA repair is an important endpoint measurement when studying the biochemical mechanisms of the DNA damage response and when investigating the efficacy of chemotherapy, which often uses DNA-damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, such methods have some limitations, which are mainly due to the lack of chromatin organization in the DNA templates used. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the corresponding damage-activated cytosolic extracts, containing biotinylated dUTP, nuclei were able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair response. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 and DDB1, stimulated UVC-induced dUTP incorporation. In contrast, a DDB2^PCNA– mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different types of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.

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在研究 DNA 损伤反应的生化机制和研究化疗（通常使用 DNA 损伤化合物）的疗效时，DNA 修复的评估是一个重要的终点测量。迄今为止，已经开发了许多体外生化表征 DNA 修复机制的方法。然而，此类方法有一些局限性，这主要是由于所使用的 DNA 模板中缺乏染色质组织。在这里，我们描述了一种功能性无细胞系统，用于研究体外DNA 修复合成，使用从经过不同基因毒性剂处理的人类细胞中分离出的 G1 期细胞核。在含有生物素化 dUTP 的相应损伤激活胞质提取物中孵育后，细胞核能够启动 DNA 修复合成。特定 DNA 合成抑制剂的使用显着减少了生物素化 dUTP 的掺入，表明修复反应的特异性。外源添加的人重组 PCNA 蛋白（但不是 UV-DNA 损伤传感器 DDB2 和 DDB1）刺激了 UVC 诱导的 dUTP 掺入。相比之下，DDB2 ^PCNA突变蛋白无法与PCNA结合，从而干扰DNA修复合成。鉴于其对不同类型 DNA 损伤的反应，该系统为研究 DNA 修复机制提供了额外的工具。

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