Substrate specificity and reaction mechanism of murine 8-oxoguanine-DNA glycosylase
Substrate specificity and reaction mechanism of murine 8-oxoguanine-DNA glycosylase
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DOI：
10.1074/jbc.m002441200
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发表时间：
2000-09-15
影响因子：
4.8
通讯作者：
Grollman, AP
中科院分区：
文献类型：
--
作者：
Zharkov, DO;Rosenquist, TA;Grollman, AP
基因组DNA容易被活性氧氧化。DNA氧化的主要产物是错误编码的碱基8-氧鸟嘌呤(8-oxoG)。8-oxoG对哺乳动物细胞的诱变作用是由8-oxoguanine-DNA糖基酶(Ogg1)、腺嘌呤-DNA糖基酶和8-oxo-dGTP酶组成的DNA修复系统阻止的。我们已经克隆、过表达并鉴定了小鼠ogg1基因的产物mOgg1。MOgg1是一种DNA糖基酶/AP裂解酶，属于核酸内切酶III家族的DNA修复酶。MOgg1的AP裂解酶活性显著低于其糖基酶活性。MOgg1与C、T或G配对时从DNA中释放8-oxoG，但有效的DNA链切割只有在8-oxoG：C的情况下才能观察到。与其他错配不同，mOgg1与含有8-oxoG：C的寡核苷酸结合很强(K-D=51.5 nM)。MOgg1在含有8-oxoG：C的底物上的平均停留时间为18.3min；NaBH4敏感中间体的积累过程表明反应机理为两步反应。测试了各种8-oxoG类似物作为mOgg1的底物。为了有效地结合mOgg1，CS上的电子抽离或氢键受体部分是必需的。裂解需要C6上的取代基和C8上的酮基。所提出的8-oxoG切除机制涉及O-8的质子化或脱氧核糖氧基。
Genomic DNA is prone to oxidation by reactive oxygen species. A major product of DNA oxidation is the miscoding base 8-oxoguanine (8-oxoG). The mutagenic effects of 8-oxoG in mammalian cells are prevented by a DNA repair system consisting of 8-oxoguanine-DNA glycosylase (Ogg1), adenine-DNA glycosylase, and 8-oxo-dGTPase. We have cloned, overexpressed, and characterized mOgg1, the product of the murine ogg1 gene. mOgg1 is a DNA glycosylase/AP lyase belonging to the endonuclease III family of DNA repair enzymes. The AP lyase activity of mOgg1 is significantly lower than its glycosylase activity. mOgg1 releases 8-oxoG from DNA when paired with C, T, or G, but efficient DNA strand nicking is observed only with 8-oxoG:C. Binding of mOgg1 to oligonucleotides containing 8-oxoG:C is strong (K-D = 51.5 nM), unlike other mispairs. The average residence time for mOgg1 bound to substrate containing 8-oxoG:C is 18.3 min; the time course for accumulation of the NaBH4-sensitive intermediate suggests a two-step reaction mechanism. Various analogs of 8-oxoG were tested as substrates for mOgg1. An electron-withdrawing or hydrogen bond acceptor moiety at CS is required for efficient binding of mOgg1. A substituent at C6 and a keto group at C8 are required for cleavage. The proposed mechanism of 8-oxoG excision involves protonation of O-8 Or the deoxyribose oxygen moiety.