A novel single-cell method provides direct evidence of persistent DNA damage in senescent cells and aged mammalian tissues.
A novel single-cell method provides direct evidence of persistent DNA damage in senescent cells and aged mammalian tissues.
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DOI：
10.1111/acel.12573
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发表时间：
2017-04
期刊：
影响因子：
7.8
通讯作者：
d'Adda di Fagagna F
中科院分区：
文献类型：
--
作者：
Galbiati A;Beauséjour C;d'Adda di Fagagna F
DNA 损伤反应 (DDR) 会阻止细胞周期进程，直到 DNA 损伤（例如 DNA 双链断裂 (DSB)）得到修复。细胞中 DSB 的存在通常通过间接技术来检测，这些技术依赖于 DSB 上蛋白质的积累，作为 DDR 的一部分。这种检测可能存在偏差，因为某些因素及其修改可能无法反映物理 DNA 损伤。 DSB 检测工具对 DDR 标记的依赖留下了一些未解答的问题。特别是，众所周知，衰老细胞表现出持久的 DDR 焦点，我们和其他人提出它是持久的 DSB，对内源性 DNA 修复活性具有抵抗力。其他人提出，这些特殊的 DDR 焦点可能不是 DNA 本身受损的位点，而是稳定的染色质修饰，称为 DNA-SCARS。在这里，我们开发了一种名为“DNA 原位连接损伤后邻近连接测定”(DI-PLA) 的方法，用于细胞中 DSB 的检测和成像。 DI-PLA 基于通过连接至生物素化的双链 DNA 寡核苷酸原位捕获固定细胞中的游离 DNA 末端，随后该寡核苷酸被抗生物素抗体识别。 PLA 与 DSB 的合作伙伴 DDR 标记一起增强了检测能力。我们通过证明 DI-PLA 能够检测培养细胞和组织中各种基因毒性损伤诱导的 DSB 来验证 DI-PLA。最重要的是，通过 DI-PLA，我们证明了培养物中的衰老细胞和衰老哺乳动物的组织都保留了与 DDR 标记相关的真正未修复的 DSB。
The DNA damage response (DDR) arrests cell cycle progression until DNA lesions, like DNA double‐strand breaks (DSBs), are repaired. The presence of DSBs in cells is usually detected by indirect techniques that rely on the accumulation of proteins at DSBs, as part of the DDR. Such detection may be biased, as some factors and their modifications may not reflect physical DNA damage. The dependency on DDR markers of DSB detection tools has left questions unanswered. In particular, it is known that senescent cells display persistent DDR foci, that we and others have proposed to be persistent DSBs, resistant to endogenous DNA repair activities. Others have proposed that these peculiar DDR foci might not be sites of damaged DNA per se but instead stable chromatin modifications, termed DNA‐SCARS. Here, we developed a method, named ‘DNA damage in situ ligation followed by proximity ligation assay’ (DI‐PLA) for the detection and imaging of DSBs in cells. DI‐PLA is based on the capture of free DNA ends in fixed cells in situ, by ligation to biotinylated double‐stranded DNA oligonucleotides, which are next recognized by antibiotin anti‐bodies. Detection is enhanced by PLA with a partner DDR marker at the DSB. We validated DI‐PLA by demonstrating its ability to detect DSBs induced by various genotoxic insults in cultured cells and tissues. Most importantly, by DI‐PLA, we demonstrated that both senescent cells in culture and tissues from aged mammals retain true unrepaired DSBs associated with DDR markers.