Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells.
Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells.
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DOI：
10.1093/nar/gkt524
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发表时间：
2013-09
影响因子：
14.9
通讯作者：
Spivak G
中科院分区：
文献类型：
--
作者：
Guo J;Hanawalt PC;Spivak G
DNA中的氧化碱基与癌症、衰老和神经退行性疾病有关。我们开发了一种将单细胞凝胶电泳（彗星）与荧光原位杂交（FISH）相结合的方法，该方法能够比较定量确定的核苷酸序列的相应链和整个基因组中低的、生理相关水平的DNA损伤。我们已经合成了单链探针靶向的目的DNA片段的末端，使用聚合酶链反应为基础的方法。这些探针有助于在单分子水平上检测损伤，因为病变被病变特异性内切核酸酶或糖基化酶转化为DNA链断裂。为了验证我们的方法，我们已经记录了转录偶联修复的环丁烷嘧啶二聚体在共济失调毛细血管扩张症突变（ATM）基因在人成纤维细胞照射254 nm紫外线在0.1 J/m2，剂量约100倍低于通常使用的。我们的方法的高特异性和灵敏度表明，7，8-二氢-8-氧代鸟嘌呤（8-oxoG）在每兆碱基约3个病变的发生率下优先修复ATM基因的转录链。我们还证明了hOGG 1，XPA，CSB和UVSSA蛋白，以及积极延长RNA聚合酶II，是这个过程所必需的，这表明DNA修复途径之间的串扰。
Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.
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影响因子：
14.9
作者：
Kitsera N;Stathis D;Lühnsdorf B;Müller H;Carell T;Epe B;Khobta A
通讯作者：
Khobta A
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作者：
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Dogliotti, E.
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Epe, B
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Yen, TJ
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作者：
Pascucci, Barbara;Lemma, Tiziana;D'Errico, Mariarosaria
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