In vitro DNA synthesis opposite oxazolone and repair of this DNA damage using modified oligonucleotides
In vitro DNA synthesis opposite oxazolone and repair of this DNA damage using modified oligonucleotides
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DOI：
10.1093/nar/28.7.1555
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发表时间：
2000-04-01
影响因子：
14.9
通讯作者：
Cadet, J
中科院分区：
文献类型：
--
作者：
Duarte, V;Gasparutto, D;Cadet, J
这项工作的重点是评估 2,2,4-三氨基恶唑酮的生物学特征，2,2,4-三氨基恶唑酮是鸟嘌呤的主要单电子和 (OH)-O-介导的氧化产物。为此，合成了两种含有独特恶唑酮残基的寡核苷酸。在此，我们报告了恶唑酮在体外 DNA 合成过程中的致突变潜力及其对 DRIA 修复酶的行为。由 Klenow 片段 exo(-) 和 Taq 聚合酶催化的与恶唑酮相反的核苷酸插入表明恶唑酮损伤主要诱导 dAMP 插入。这表明DNA中恶唑酮的形成可能导致G-->T颠换，另一方面，当用DNA聚合酶β进行DNA合成时，恶唑酮代表了一种阻断性损伤。有趣的是，用甲酰胺嘧啶DNA N-糖基化酶（Fpg）和核酸内切酶III（endo III）进行的DNA修复实验表明，恶唑酮是 两种酶的底物。 Fpg 介导的氧化鸟嘌呤损伤去除的 k(cat)/K-m 值表明，8-oxo-7,8-二氢鸟嘌呤仅是比恶唑酮稍好的底物。在 endo III 介导的修饰碱基裂解的情况下，目前的结果表明，恶唑酮是比 5-OHC（一种氧化嘧啶碱基）更好的底物。最后，对 Fpg 消化含恶唑酮的寡核苷酸时释放的 DNA 片段进行 MALDI-TOF-MS 分析，深入了解寡核苷酸切割的酶促机制。
Emphasis was placed in this work on the assessment of biological features of 2,2,4-triaminooxazolone, a major one-electron and (OH)-O-.-mediated oxidation product of guanine, For this purpose, two oligonucleotides that contain a unique oxazolone residue were synthesized. Herein we report the mutagenic potential of oxazolone during in vitro DNA synthesis and its behavior towards DRIA repair enzymes. Nucleotide insertion opposite oxazolone, catalyzed by Klenow fragment exo(-) and Taq polymerase indicates that the oxazolone lesion induces mainly dAMP insertion. This suggests that the formation of oxazolone in DNA may lead to G-->T transversions, On the other hand, oxazolone represents a blocking lesion when DNA synthesis is performed with DNA polymerase beta, Interestingly, DNA repair experiments carried out with formamidopyrimidine DNA N-glycosylase (Fpg) and endonuclease III (endo III) show that oxazolone is a substrate for both enzymes. Values of k(cat)/K-m for the Fpg-mediated removal of oxidative guanine lesions revealed that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than oxazolone, In the case of endo III-mediated cleavage of modified bases, the present results suggest that oxazolone is a better substrate than 5-OHC, an oxidized pyrimidine base. Finally, MALDI-TOF-MS analysis of the DNA fragments released upon digestion of an oxazolone-containing oligonucleotide by Fpg gave insights into the enzymatic mechanism of oligonucleotide cleavage.