Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling.
Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling.
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DOI：
10.3389/fgene.2013.00135
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发表时间：
2013
影响因子：
3.7
通讯作者：
Leitenstorfer A
中科院分区：
文献类型：
--
作者：
Ferrando-May E;Tomas M;Blumhardt P;Stöckl M;Fuchs M;Leitenstorfer A
我们对高等真核生物DNA损伤反应机制的理解，在很大程度上取决于我们对维持基因组完整性的细胞机制的时间和空间组织的剖析能力。为了实现这一目标，我们需要实验工具在预先定义的位置以高空间精度造成DNA损伤，并以足够的时间分辨率可视化随后的反应。近红外飞秒激光脉冲通过高级扫描显微镜的高孔径物镜聚焦，提供了在亚核尺寸的3d受限体积中诱导DNA损伤的优势。这种高空间分辨率源于激励过程的高度非线性性质。本文综述了基于近红外超快激光器广泛可调谐和用户友好技术的最新进展。与目前普遍使用的UV或VIS激光照射（后者与光敏剂结合使用）相比，我们对这种方法进行了DNA微损伤的关键评估。概述了当前和未来在DNA修复和DNA损伤依赖的染色质动力学领域的应用。最后讨论了适当的仿真和定量建模的要求。我们特别关注测量DNA损伤对核蛋白迁移率影响的方法，并考虑FRAP和光激活常用分析模型的优缺点及其在非线性光扰动实验中的适用性。
Our understanding of the mechanisms governing the response to DNA damage in higher eucaryotes crucially depends on our ability to dissect the temporal and spatial organization of the cellular machinery responsible for maintaining genomic integrity. To achieve this goal, we need experimental tools to inflict DNA lesions with high spatial precision at pre-defined locations, and to visualize the ensuing reactions with adequate temporal resolution. Near-infrared femtosecond laser pulses focused through high-aperture objective lenses of advanced scanning microscopes offer the advantage of inducing DNA damage in a 3D-confined volume of subnuclear dimensions. This high spatial resolution results from the highly non-linear nature of the excitation process. Here we review recent progress based on the increasing availability of widely tunable and user-friendly technology of ultrafast lasers in the near infrared. We present a critical evaluation of this approach for DNA microdamage as compared to the currently prevalent use of UV or VIS laser irradiation, the latter in combination with photosensitizers. Current and future applications in the field of DNA repair and DNA-damage dependent chromatin dynamics are outlined. Finally, we discuss the requirement for proper simulation and quantitative modeling. We focus in particular on approaches to measure the effect of DNA damage on the mobility of nuclear proteins and consider the pros and cons of frequently used analysis models for FRAP and photoactivation and their applicability to non-linear photoperturbation experiments.