O'Connor T R, Graves R J, de Murcia G, Castaing B, Laval J
Groupe Réparation des Lésions Radio-et chimio-induites, Centre National de la Recherche Scientifique URA147/Institut National de la Santé et de la Recherche Médicale U140, Institut Gustave-Roussy, Villejuif, France.
J Biol Chem. 1993 Apr 25;268(12):9063-70.
The Fpg protein of Escherichia coli is a DNA repair enzyme with DNA glycosylase, abasic site nicking, and deoxyribose excising activities. Analysis of the amino acid sequence of this protein suggests that the Fpg protein is a zinc finger protein with a Cys-X2-Cys-X16-Cys-X2-Cys motif. Competition experiments show that the Fpg protein substitutes Cu(II), Cd(II), and Hg(II), metal ions classically associated with substitutions in zinc finger proteins. The Fpg protein activities are inhibited following the reaction with a Cys-specific reagent at low protein:reagent ratios, suggesting that these residues are important for the enzymatic activities. Site-directed mutagenesis was used to produce 6 mutant Fpg proteins with Cys-->Gly mutations. Substitution of the zinc in these proteins by 65Zn(II) indicates that all the proteins bind zinc, but the Zn(II) is not retained as strongly in the zinc finger mutants. The mutations in the Fpg protein outside the zinc finger consensus sequence do not eliminate the Fapy-DNA glycosylase and abasic site nicking. One of the Fpg mutant proteins outside the zinc finger has a reduced capacity to release deoxyribose from abasic sites. Cys-->Gly mutations in the zinc finger consensus sequence reduce all three aforementioned activities substantially. The purified Fpg proteins with Cys-->Gly mutations in the zinc finger consensus sequence do not incise DNA at abasic sites with the same efficiency nor mechanism as the native Fpg protein. The wild type Fpg protein and the Fpg proteins mutated outside the zinc finger sequence bind an oligonucleotide with a unique chemically reduced abasic site in a defined sequence as assayed by retention on nitrocellulose filters, whereas the mutant Fpg proteins within the zinc finger sequence do not bind to the same oligonucleotide. Therefore, the disruption of zinc coordination in the zinc finger of the Fpg protein is associated with decreased binding capacity to DNA as well as decreased enzymatic activities.
大肠杆菌的Fpg蛋白是一种具有DNA糖基化酶、无碱基位点切口和脱氧核糖切除活性的DNA修复酶。对该蛋白氨基酸序列的分析表明，Fpg蛋白是一种具有Cys-X2-Cys-X16-Cys-X2-Cys基序的锌指蛋白。竞争实验表明，Fpg蛋白能替代锌指蛋白中经典的与替代相关的Cu(II)、Cd(II)和Hg(II)金属离子。在低蛋白与试剂比例下，Fpg蛋白与半胱氨酸特异性试剂反应后其活性受到抑制，这表明这些残基对酶活性很重要。定点诱变用于产生6种具有Cys→Gly突变的Fpg突变蛋白。用65Zn(II)替代这些蛋白中的锌表明，所有蛋白都能结合锌，但锌指突变体中Zn(II)的保留能力不强。Fpg蛋白在锌指共有序列之外的突变不会消除Fapy-DNA糖基化酶和无碱基位点切口。锌指之外的一种Fpg突变蛋白从无碱基位点释放脱氧核糖的能力降低。锌指共有序列中的Cys→Gly突变显著降低了上述三种活性。在锌指共有序列中具有Cys→Gly突变的纯化Fpg蛋白在无碱基位点切割DNA的效率和机制与天然Fpg蛋白不同。通过硝酸纤维素滤膜上的保留分析，野生型Fpg蛋白和在锌指序列之外突变的Fpg蛋白能结合具有特定化学还原无碱基位点的寡核苷酸，而锌指序列内的突变Fpg蛋白则不与同一寡核苷酸结合。因此，Fpg蛋白锌指中锌配位的破坏与DNA结合能力下降以及酶活性降低有关。
