Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; Doctoral School of Exact and Natural Sciences, Jagiellonian University, Kraków, Poland.
Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.
J Photochem Photobiol B. 2024 Oct;259:113001. doi: 10.1016/j.jphotobiol.2024.113001. Epub 2024 Aug 2.
Green fluorescent protein (GFP) has opened vast new avenues in studies of live cells and is generally perceived as a benign, nontoxic and harmless fluorescent tag. We demonstrat that excited GFP is capable of inducing substantial DNA damage in cells expressing fusion proteins. In the presence of GFP, even low doses of blue light (12 μJ) induce single strand breaks (SSBs). When the fluorescence of GFP located in the cell nucleus or in the cytoplasm is excited by a much higher dose (17 mJ), DNA double-strand breaks (DSBs) are also induced. Such breaks are induced even when GFP is placed and illuminated in culture medium outside of living cells. We demonstrate that DNA damage is induced by singlet oxygen, which is generated by excited GFP. Although short exposures of live cells to exciting light typically used in fluorescence microscopy induce SSBs but carry little risk of inducing DNA double-strand breaks, larger doses, which may be used in FRAP, FLIM, FCS and super-resolution fluorescence microscopy studies, are capable of inducing not only numerous SSBs but also DSBs.
绿色荧光蛋白 (GFP) 在活细胞研究中开辟了广阔的新途径，通常被认为是一种良性、无毒和无害的荧光标记物。我们证明，激发 GFP 能够诱导表达融合蛋白的细胞产生大量 DNA 损伤。在 GFP 的存在下，即使是低剂量的蓝光（12 μJ）也会诱导单链断裂（SSBs）。当位于细胞核或细胞质中的 GFP 荧光被更高剂量（17 mJ）激发时，也会诱导 DNA 双链断裂（DSBs）。即使 GFP 放置在活细胞外的培养基中并被照射，也会发生这种断裂。我们证明，DNA 损伤是由激发态 GFP 产生的单线态氧引起的。虽然活细胞暴露于荧光显微镜中常用的激发光通常会诱导 SSBs，但很少有诱导 DNA 双链断裂的风险，但更大的剂量，可能用于 FRAP、FLIM、FCS 和超分辨率荧光显微镜研究，不仅能够诱导大量的 SSBs，还能够诱导 DSBs。
