Department of Biomedical Engineering, School of Engineering, University of California, Irvine, Irvine, CA 92697.
Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697.
Mol Biol Cell. 2019 Sep 15;30(20):2584-2597. doi: 10.1091/mbc.E18-10-0650. Epub 2019 Aug 7.
DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal the novel prosurvival response to PARP activation through a change in cellular metabolism and demonstrate how unique applications of advanced fluorescence imaging and laser microirradiation-induced DNA damage can be a powerful tool to interrogate damage-induced metabolic changes at high spatiotemporal resolution in a live cell.
DNA 损伤信号对于维持基因组完整性和细胞命运决策至关重要。聚（ADP-核糖）聚合酶 1（PARP1）是一种 DNA 损伤传感器，它能够快速且具有损伤剂量和复杂性依赖性地被激活，在损伤部位的初始染色质组织和 DNA 修复途径选择中发挥关键作用。然而，我们对其在受损细胞中激活的全细胞后果的理解仍然有限。使用荧光寿命成像显微镜的相子方法和荧光生物传感器，并结合激光微照射，我们发现，在细胞核 DNA 损伤的情况下，NADH 结合分数会迅速增加，这是由 PARP 依赖性的 NAD+耗竭引发的。这种变化与代谢平衡向氧化磷酸化（oxphos）而非糖酵解的转变有关。抑制 oxphos，但不是糖酵解，会导致由于 PARP 依赖性的 ATP 耗竭而导致的 parthanatos，这表明 oxphos 对受损细胞的存活变得至关重要。这些结果揭示了通过改变细胞代谢对 PARP 激活的新的生存反应，并展示了如何通过先进的荧光成像和激光微照射诱导的 DNA 损伤的独特应用，以高时空分辨率在活细胞中研究损伤诱导的代谢变化。
