Psoralen conjugates for visualization of genomic interstrand cross-links localized by laser photoactivation
Psoralen conjugates for visualization of genomic interstrand cross-links localized by laser photoactivation
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DOI：
10.1021/bc060309t
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发表时间：
2007-03-01
影响因子：
4.7
通讯作者：
Seidman, Michael M.
中科院分区：
文献类型：
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作者：
Thazhathveetil, Arun Kalliat;Liu, Su-Ting;Seidman, Michael M.
DNA链间交联由化疗药物以及正常氧化代谢的副产物形成。尽管它们的重要性，交联代谢的途径知之甚少。激光共聚焦显微镜已成为研究DNA损伤修复的有力工具，可以通过免疫荧光试剂检测。为了将这种方法应用于交联修复，我们合成了4，5 '，8-三甲基胡椒烯（TMP）和容易检测的化合物如丽丝胺罗丹明B磺酰氯（LRB-SC）、生物素和地高辛的缀合物。这些共轭物被激活的UVA，我们已经分析了细胞内定位的DNA损伤和DNA反应的共聚焦和免疫荧光显微镜。LRB-SC-TMP偶联物2主要出现在线粒体中，而生物素-TMP偶联物4优先定位于细胞质中。UVA与TMP 7a和4，5 '-二甲基当归素（DMA）7 b的地高辛缀合物形成的加合物（仅形成单加合物）主要定位于细胞核。将与7a和7 b一起孵育的细胞暴露于指向所关注的限定核区域的364 nm UV激光导致局部加合物形成，其可以通过免疫荧光可视化。修复能力强的细胞能够去除光加合物，而修复缺陷的细胞则无法修复损伤。结果表明，地高辛-TMP偶联物7a和地高辛-DMA偶联物7 b可用于研究激光定位的DNA单加合物和交联的修复。
DNA interstrand cross-links are formed by chemotherapy drugs as well as by products of normal oxidative metabolism. Despite their importance, the pathways of cross-link metabolism are poorly understood. Laser confocal microscopy has become a powerful tool for studying the repair of DNA lesions that can be detected by immunofluorescent reagents. In order to apply this approach to cross-link repair, we have synthesized conjugates of 4,5',8-trimethylpsoralen (TMP) and easily detected compounds such as Lissamine rhodamine B sulfonyl chloride (LRB-SC), biotin, and digoxigenin. These conjugates are activated by UVA, and we have analyzed the intracellular localization of DNA damage and DNA reactivity by confocal and immunofluorescence microscopy. The LRB-SC-TMP conjugate 2 appeared mainly in the mitochondria, while the biotin-TMP conjugate 4 preferentially localized in the cytoplasm. Adducts formed by UVA and digoxigenin conjugates of TMP 7a and 4,5'-dimethylangelicin (DMA) 7b, which forms only monoadducts, were largely localized to the nucleus. Exposure of cells incubated with 7a and 7b to a 364 nm UV laser directed toward defined nuclear regions of interest resulted in localized adduct formation which could be visualized by immunofluorescence. Repair-proficient cells were able to remove the photoadducts, while repair-deficient cells were unable to repair the damage. The results indicated that the digoxigenin-TMP conjugate 7a and digoxigenin-DMA conjugate 7b can be used for studying the repair of laser localized DNA monoadducts and cross-links.