DNA Multiphoton Absorption Generates Localized Damage for Studying Repair Dynamics in Live Cells
DNA Multiphoton Absorption Generates Localized Damage for Studying Repair Dynamics in Live Cells
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DOI：
10.1016/j.bpj.2011.09.031
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发表时间：
2011-11-02
影响因子：
3.4
通讯作者：
Fecko, Christopher J.
中科院分区：
文献类型：
--
作者：
Daddysman, Matthew K.;Fecko, Christopher J.
利用活细胞成像的DNA修复的时空动力学的调查是由在光学显微镜中产生良好定义的区域的紫外线photolisions的能力。我们证明，在活细胞中的DNA与可见的飞秒脉冲的多光子激发产生胸腺嘧啶环嘧啶二聚体（CPD），主要的紫外线DNA的光产物。使用波长范围为至少400至525 nm的脉冲产生具有立方到超立方功率依赖性的CPD。我们表明，CPD被限制在所有三个空间维度，使多光子激发的DNA与可见光的理想技术，用于产生本地化的DNA photolisions在各种各样的样品，从培养的细胞到较厚的组织。我们证明了这种方法的实用性，将其应用于调查的时空招聘GFP标记的拓扑异构酶I（TopI）的光学厚果蝇唾液腺活细胞内的多线染色体中的局部DNA损伤的网站。
Investigations into the spatiotemporal dynamics of DNA repair using live-cell imaging are aided by the ability to generate well defined regions of ultravioletlike photolesions in an optical microscope. We demonstrate that multiphoton excitation of DNA in live cells with visible femtosecond pulses produces thymine cyclopyrimidine dinners (CPDs), the primary ultraviolet DNA photoproduct. The CPDs are produced with a cubic to supercubic power dependence using pulses in the wavelength range from at least 400 to 525 nm. We show that the CPDs are confined in all three spatial dimensions, making multiphoton excitation of DNA with visible light an ideal technique for generating localized DNA photolesions in a wide variety of samples, from cultured cells to thicker tissues. We demonstrate the utility of this method by applying it to investigate the spatiotemporal recruitment of GFP-tagged topoisomerase I (TopI) to sites of localized DNA damage in polytene chromosomes within live cells of optically thick Drosophila salivary glands.