Muster Britta, Rapp Alexander, Cardoso M Cristina
Cell Biology and Epigenetics, Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany.
AIMS Genet. 2017 Feb 21;4(1):47-68. doi: 10.3934/genet.2017.1.47. eCollection 2017.
Laser micro-irradiation can be used to induce DNA damage with high spatial and temporal resolution, representing a powerful tool to analyze DNA repair in the context of chromatin. However, most lasers induce a mixture of DNA damage leading to the activation of multiple DNA repair pathways and making it impossible to study individual repair processes. Hence, we aimed to establish and validate micro-irradiation conditions together with inhibition of several key proteins to discriminate different types of DNA damage and repair pathways using lasers commonly available in confocal microscopes. Using time-lapse analysis of cells expressing fluorescently tagged repair proteins and also validation of the DNA damage generated by micro-irradiation using several key damage markers, we show that irradiation with a 405 nm continuous wave laser lead to the activation of all repair pathways even in the absence of exogenous sensitization. In contrast, we found that irradiation with 488 nm laser lead to the selective activation of non-processive short-patch base excision and single strand break repair, which were further validated by PARP inhibition and metoxyamine treatment. We conclude that these low energy conditions discriminated against processive long-patch base excision repair, nucleotide excision repair as well as double strand break repair pathways.
激光微照射可用于以高空间和时间分辨率诱导DNA损伤，是在染色质背景下分析DNA修复的有力工具。然而，大多数激光会诱导多种DNA损伤，导致多种DNA修复途径被激活，从而无法研究单个修复过程。因此，我们旨在建立并验证微照射条件，并抑制几种关键蛋白，以便使用共聚焦显微镜中常用的激光来区分不同类型的DNA损伤和修复途径。通过对表达荧光标记修复蛋白的细胞进行延时分析，并使用几种关键损伤标记物验证微照射产生的DNA损伤，我们发现，即使在没有外源性增敏的情况下，用405 nm连续波激光照射也会导致所有修复途径被激活。相比之下，我们发现用488 nm激光照射会导致非连续短片段碱基切除和单链断裂修复的选择性激活，这通过PARP抑制和甲氧基胺处理得到了进一步验证。我们得出结论，这些低能量条件区分了连续长片段碱基切除修复、核苷酸切除修复以及双链断裂修复途径。
