CLEAVAGE AND BINDING OF A DNA FRAGMENT CONTAINING A SINGLE 8-OXOGUANINE BY WILD-TYPE AND MUTANT FPG PROTEINS
CLEAVAGE AND BINDING OF A DNA FRAGMENT CONTAINING A SINGLE 8-OXOGUANINE BY WILD-TYPE AND MUTANT FPG PROTEINS
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DOI：
10.1093/nar/21.12.2899
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发表时间：
1993-06-25
影响因子：
14.9
通讯作者：
BOITEUX, S
中科院分区：
文献类型：
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作者：
CASTAING, B;GEIGER, A;BOITEUX, S
利用含有单个7,8-二氢-8-氧鸟嘌呤（8-OxoG）残基的34聚寡核苷酸研究了大肠杆菌Fpg蛋白的酶学和DNA结合特性。在8-OxoG与C （*G/C）或T （*G/T）相反的双链中，Fpg蛋白对含8-OxoG链的切割率最高。相反，含有8-OxoG的双链对G （*G/G）和A （*G/A）的切口速度分别慢5倍和200倍。凝胶缓凝研究表明，Fpg蛋白对8-OxoG为相反嘧啶的双链具有较强的亲和力，而对8-OxoG为相反嘌呤的双链亲和力较弱。K(D)app值分别为0.6 nM （*G/C）、1.0 nM （*G/T）、6.0 nM （*G/G）和16.0 nM （*G/A）。Fpg蛋白也与未修饰的（G/C）双相结合，并测量了90 nM的K(D)app。利用细菌粗裂解物研究了（*G/C）双链的裂解和结合。野生型大肠杆菌粗提物切割含有8-OxoG的链，形成具有（*G/C）双链的特异性阻滞复合物。这两种反应是由Fpg蛋白介导的，因为它们没有从Fpg基因灭活的菌株的粗提取物中观察到。此外，我们还研究了6个具有Cys -> Gly突变的Fpg蛋白的特性。结果表明，锌指序列外Cys -> Gly突变的2个Fpg蛋白切割含有8-OxoG的链，与（*G/C）双工形成复合物，抑制了Fpg -1突变体的突变表型。相比之下，在锌指基序中具有Cys -> Gly突变的4种Fpg蛋白既不能切割也不能结合（*G/C）双链，也不能抑制Fpg -1突变表型。
A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding properties of the Fpg protein from E.coli. The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for duplexes where 8-OxoG was opposite C (*G/C) or T (*G/T). In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (*G/G) and A (*G/A) were 5-fold and 200-fold slower. Gel retardation studies showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines. K(D)app values were 0.6 nM (*G/C), 1.0 nM (*G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A). The Fpg protein also binds to unmodified (G/C) duplex and a K(D)app of 90 nM was measured. The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates. Wild type E.coli crude extract incised the 8-OxoG containing strand and formed a specific retardation complex with the (*G/C) duplex. These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial strain whose fpg gene was inactivated. Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys --> Gly mutations. The results showed that the 2 Fpg proteins with Cys --> Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (*G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant. In contrast, the 4 Fpg proteins with Cys --> Gly mutations within the zinc finger motif neither cleave nor bind the (*G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotype.