Substrate specificity of Fpg protein. Recognition and cleavage of oxidatively damaged DNA.
Substrate specificity of Fpg protein. Recognition and cleavage of oxidatively damaged DNA.
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发表时间：
1994-05
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通讯作者：
Julia Tchou;V. Bodepudi;S. Shibutani;I. Antoshechkin;Jeffrey H. Miller;A. Grollman;F. Johnson
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作者：
Julia Tchou;V. Bodepudi;S. Shibutani;I. Antoshechkin;Jeffrey H. Miller;A. Grollman;F. Johnson
大肠杆菌的8-氧代鸟嘌呤-DNA糖基化酶，也称为甲酰氨基嘧啶-DNA糖基化酶（Fpg蛋白），具有N-糖基化酶和AP-裂解酶活性。这种酶通过有效地从DNA中去除甲酰胺基嘧啶损伤和8-氧代鸟嘌呤残基来修复氧化DNA损伤。使用含有各种8-氧代嘌呤的定义的寡脱氧核苷酸来检查Fpg蛋白的底物特异性，并确定DNA中的官能团在损伤识别和催化中的作用。对含有8-氧代-2 '-脱氧鸟嘌呤、8-氧代-2'-脱氧腺嘌呤、8-氧代-2 '-脱氧水云碱、8-氧代-2'-脱氧肌苷、脱碱基位点和C8-氨基芴鸟嘌呤的开环加合物的双链寡脱氧核苷酸建立Fpg蛋白的结合亲和力。8-oxodG：dC的C8酮基存在于大沟中，并与紧密结合相关（Kd = 8.9 nM）。当C8酮官能团位于小沟中时，结合弱得多，如8-oxodG：dA（Kd = 340 nM）。测定裂解反应的Km和Vmax。对于含有C6和C8酮基的8-氧代嘌呤的寡脱氧核苷酸双链体，特异性常数（Kcat/Km）始终较高，如8-oxodG：dC和8-oxodI：dC，其中Kcat/Km分别为9.3和18 min-1 nM x 10（-3）。8-oxodN：dC缺少C6酮基;特异性常数为0.024 min-1 nM x 10（-3）。两者合计，我们的数据表明，C8酮基的8-oxodeoxyguanine和羰基部分的formamidopyrimidine使Fpg蛋白识别和结合双链体DNA含有这些修改的碱基。涉及底物的C6酮基的酶催化反应导致这些病变的去除。一种机制，涉及质子化在O-6的8-氧代鸟嘌呤提出帐户的N-糖基化酶活性的这种酶。
The 8-oxoguanine-DNA glycosylase of Escherichia coli, also known as formamidopyrimidine-DNA glycosylase (Fpg protein), has N-glycosylase and AP-lyase activities. This enzyme repairs oxidative DNA damage by efficiently removing formamidopyrimidine lesions and 8-oxoguanine residues from DNA. Defined oligodeoxynucleotides containing various 8-oxopurines were used to examine the substrate specificity of Fpg protein and to establish the role of functional groups in DNA on damage recognition and catalysis. Binding affinities of Fpg protein were established for duplex oligodeoxynucleotides containing 8-oxo-2'-deoxyguanine, 8-oxo-2'-deoxyadenine, 8-oxo-2'-deoxynebularine, 8-oxo-2'-deoxyinosine, abasic sites, and a ring-open adduct of C8-aminofluorene guanine. The C8 keto group of 8-oxodG:dC presents in the major groove and is correlated with tight binding (Kd = 8.9 nM). Binding is much weaker when the C8 keto functional group is in the minor groove, as in 8-oxodG:dA (Kd = 340 nM). Km and Vmax were determined for the cleavage reaction. Specificity constants (Kcat/Km) are consistently higher for oligodeoxynucleotide duplexes containing 8-oxopurines with C6 and C8 keto groups, as in 8-oxodG:dC and 8-oxodI:dC, where Kcat/Km are 9.3 and 18 min-1 nM x 10(-3), respectively. 8-oxodN:dC lacks the C6 keto group; the specificity constant is 0.024 min-1 nM x 10(-3). Taken together, our data suggest that the C8 keto group of 8-oxodeoxyguanine and the carbonyl moiety of formamidopyrimidine enable Fpg protein to recognize and bind duplex DNA containing these modified bases. An enzyme-catalyzed reaction involving the C6 keto group of the substrate leads to removal of these lesions. A mechanism involving protonation at O-6 of 8-oxoguanine is proposed to account for the N-glycosylase activity of this enzyme.