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首页> 美国卫生研究院文献>other >Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry
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Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

机译：气相色谱-负化学电离-质谱法测量培养的人细胞中外源5-羟甲基-2-脱氧尿苷的掺入和修复

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摘要

The DNA of all organisms is constantly damaged by oxidation. Among the array of damage products is 5-hydroxymethyluracil, derived from oxidation of the thymine methyl group. Previous studies have established that HmU can be a sensitive and valuable marker of DNA damage. More recently, the corresponding deoxynucleoside, 5-hydroxymethyl-2′-deoxyuridine (HmdU) has proven to be valuable for the introduction of controlled amounts of a single type of damage lesion into the DNA of replicating cells, which is subsequently repaired by the base excision repair pathway. Complicating the study of HmU formation and repair, however, is the known chemical reactivity of the hydroxymethyl group of HmU under conditions used to hydrolyze DNA. In the work reported here, this chemical property has been exploited by creating conditions that convert HmU to the corresponding methoxymethyluracil (MmU) derivative that can be further derivatized to the 3,5-bis-(trifluoromethyl)benzyl analog. This derivatized compound can be detected by gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) with good sensitivity. Using isotopically enriched exogenous HmdU and human osteosarcoma cells (U2OS) in culture, we demonstrate that this method allows for the measurement of HmU in DNA formed from incorporation of exogenous HmdU. We further demonstrate that addition of isotopically enriched uridine to the culture medium allows for the simultaneous measurement of DNA replication and repair kinetics. This sensitive and facile method should prove valuable for studies on DNA oxidation damage and repair in living cells.
机译：所有生物的DNA都会不断被氧化破坏。在一系列损伤产物中是衍生自胸腺嘧啶甲基的氧化的5-羟甲基尿嘧啶。先前的研究已经确定，HmU可以作为DNA损伤的敏感且有价值的标记。最近，事实证明，相应的脱氧核苷5-羟甲基-2'-脱氧尿苷（HmdU）对于将可控制量的单一类型损伤灶引入复制细胞的DNA中是很有价值的，然后再由碱基修复切除修复途径。然而，在用于水解DNA的条件下，HmU的羟甲基的已知化学反应性使HmU形成和修复的研究复杂化。在本文报道的工作中，通过创造条件将HmU转化为相应的甲氧基甲基尿嘧啶（MmU）衍生物，可以进一步衍生化为3,5-双-（三氟甲基）苄基类似物，从而利用了这种化学性质。通过气相色谱-负化学电离质谱（GC-NCI-MS）可以很好地检测到这种衍生化的化合物。在文化中使用同位素富集的外源性HmdU和人骨肉瘤细胞（U2OS），我们证明了该方法可用于测量由外源性HmdU掺入形成的DNA中的HmU。我们进一步证明，向培养基中添加同位素富集的尿苷可以同时测量DNA复制和修复动力学。这种灵敏，简便的方法对于活细胞DNA氧化损伤和修复的研究应具有重要的价值。

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