Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage
Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage
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DOI：
10.1093/nar/24.3.418
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发表时间：
1996-02-01
影响因子：
14.9
通讯作者：
Krokan, HE
中科院分区：
文献类型：
--
作者：
Dizdaroglu, M;Karakaya, A;Krokan, HE
尿嘧啶DNA N-糖基化酶是一种从DNA中释放尿嘧啶的修复酶，该酶的主要功能可能是保护基因组免受DNA中胞嘧啶脱氨基产生的预诱变尿嘧啶的影响。在此，我们报告了人尿嘧啶DNA N-糖基化酶也识别三种尿嘧啶衍生物，它们是DNA中胞嘧啶通过羟基自由基攻击或其他氧化过程产生的主要产物，通过在充气水溶液中对DNA进行γ-照射来制备DNA底物，并与人尿嘧啶DNA N-糖基化酶、热灭活酶或缓冲液一起孵育。用气相色谱/同位素稀释质谱法对衍生化后的上清和水解衍生化后的沉淀进行分析。结果表明，人尿嘧啶DNA N-糖基化酶从DNA上切割异二尿酸、5-羟基尿嘧啶和四氧嘧啶的表观Km值分别为530、450和660 nM，这些尿嘧啶类似物的切除与最近描述的人尿嘧啶DNA N-糖基化酶识别尿嘧啶的机制一致[Mol，C.D.，Arval，A，S.，Slupphaug，G.，Kavil，B.，阿尔赛斯湖，克罗坎阁下和Tainer，J.A.（1995）Cell，80，869-878]，在DNA样品中鉴定的其他九种嘧啶和嘌呤衍生产物不是该酶的底物。结果表明，人尿嘧啶DNA N-糖基化酶可能具有修复氧化DNA损伤的功能。
Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA, A major function of this enzyme is presumably to protect the genome from pre-mutagenic uracil resulting from deamination of cytosine in DNA, Here, we report that human uracil DNA N-glycosylase also recognizes three uracil derivatives that are generated as major products of cytosine in DNA by hydroxyl radical attack or other oxidative processes, DNA substrates were prepared by gamma-irradiation of DNA in aerated aqueous solution and incubated with human uracil DNA N-glycosylase, heat-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernatant fractions were then separated, Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry, The results demonstrated that human uracil DNA N-glycosylase excised isodialuric acid, 5-hydroxyuracil and alloxan from DNA with apparent K-m values of similar to 530, 450 and 660 nM, respectively, The excision of these uracil analogues is consistent with the recently described mechanism for recognition of uracil by human uracil DNA N-glycosylase [Mol,C.D., Arval,A,S., Slupphaug,G., Kavil,B., Alseth,l., Krokan,H.E. and Tainer,J.A. (1995) Cell, 80, 869-878], Nine other pyrimidine and purine-derived products that were identified in DNA samples were not substrates for the enzyme, The results indicate that human uracil DNA N-glycosylase may have a function in the repair of oxidative DNA damage.