8-OXOGUANINE (8-HYDROXYGUANINE) DNA GLYCOSYLASE AND ITS SUBSTRATE-SPECIFICITY
8-OXOGUANINE (8-HYDROXYGUANINE) DNA GLYCOSYLASE AND ITS SUBSTRATE-SPECIFICITY
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DOI：
10.1073/pnas.88.11.4690
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发表时间：
1991-06-01
影响因子：
11.1
通讯作者：
NISHIMURA, S
中科院分区：
文献类型：
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作者：
TCHOU, J;KASAI, H;NISHIMURA, S
通过使用含有8-氧代-7，8-二氢-2 '-脱氧鸟苷（8-oxodG）、8-氧代-7，8-二氢-2'-脱氧腺苷（8-oxodA）和2，6-二氨基-4-羟基-5-（N-甲基）甲酰胺基嘧啶（Me-Fa）的单个残基的确定的双链寡脱氧核苷酸，比较FPG蛋白（也称为甲酰胺基嘧啶DNA糖基化酶）和8-羟基鸟嘌呤核酸内切酶的底物特异性。 含有与dC、dG或dT相对定位的8-oxodG的双链体被切割，而单链DNA和含有与四种DNA碱基中的任一种相对定位的8-oxodG、dA或8-oxodA的双链体是相对抗性的。 两种酶都能将含有8-oxoG.dC的双链体切割到修饰碱基的3'和5'端，但不能切割含有合成脱碱基位点、含有dG的错配或未修饰DNA的双链体DNA。 8-氧代鸟嘌呤，通过HPLC-电化学检测技术鉴定，在酶促反应过程中释放。 FPG蛋白作用于含有位于dC相对位置的单个Me-fetamine或8-oxodG残基的双链体底物的表观K（m）值分别为41和8 nM，8-羟基鸟嘌呤核酸内切酶的表观K（m）值分别为30和13 nM。 比较两种酶活性的性质表明它们是相同的。 鉴于8-oxodG在细胞DNA中的广泛分布，这种病变的表现出的错误编码和致突变性，以及存在的细菌基因编码的FPG蛋白质，我们建议，8-oxodG DNA是主要的生理底物的组成糖基化酶中发现的细菌和哺乳动物细胞。
Substrate specificities of FPG protein (also known as formamidopyrimidine DNA glycosylase) and 8-hydroxyguanine endonuclease were compared by using defined duplex oligodeoxynucleotides containing single residues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), and 2,6-diamino-4-hydroxy-5-(N-methyl)formamidopyrimidine (Me-Fapy). Duplexes containing 8-oxodG positioned opposite dC, dG, or dT were cleaved, whereas single-stranded DNA and duplexes containing 8-oxodG.dA or 8-oxodA positioned opposite any of the four DNA bases were relatively resistant. Both enzymes cut duplexes containing 8-oxoG.dC 3' and 5' to the modified base but failed to cleave duplex DNA containing synthetic abasic sites, mismatches containing dG, or unmodified DNA. 8-Oxoguanine, identified by HPLC-electrochemical detection techniques, was released during the enzymatic reaction. Apparent K(m) values for FPG protein acting on duplex substrates containing a single Me-fapy or 8-oxodG residue positioned opposite dC were 41 and 8 nM, respectively, and those for 8-hydroxyguanine endonuclease were 30 and 13 nM, respectively. Comparison of the properties of the two enzyme activities suggest that they are identical. In view of the widespread distribution of 8-oxodG in cellular DNA, the demonstrated miscoding and mutagenic properties of this lesion, and the existence of a bacterial gene coding for FPG protein, we propose that 8-oxodG DNA is the primary physiological substrate for a constituent glycosylase found in bacteria and mammalian cells.