Castaing B, Boiteux S, Zelwer C
Centre de Génétique Moléculaire, Université Pierre et Marie Curie, Gif/Yvette, France.
Nucleic Acids Res. 1992 Feb 11;20(3):389-94. doi: 10.1093/nar/20.3.389.
The E. coli Formamidopyrimidine-DNA Glycosylase (FPG protein), a monomeric DNA repair enzyme of 30.2 kDa, was purified to homogeneity in large quantities. The FPG protein excises imidazole ring-opened purines and 8-hydroxyguanine residues from DNA. Besides DNA glycosylase activity, the FPG protein is endowed with an EDTA-resistant activity which nicks DNA at apurinic/apyrimidic sites (AP sites). In contrast, DNAs containing chemically reduced AP sites are not incised by the FPG protein. However, the DNA glycosylase activity of the FPG protein is strongly inhibited in the presence of a purified synthetic 24 base-pair double-stranded oligonucleotide which contains a single apurinic site transformed chemically through borohydride reduction into a ring-opened deoxyribose derivative. The ability of the FPG protein to form a complex with this synthetically modified DNA was studied by electrophoresis in non-denaturing polyacrylamide gels. The FPG protein specifically binds the double-stranded oligonucleotide containing an apurinic site previously reduced in the presence of sodium borohydride. The complex was identified as a single retardation band on non-denaturing polyacrylamide gel electrophoresis. Complex formation is reversible and an apparent dissociation constant, KDapp, of 2.6 x 10(-10) M was determined. In contrast, no such retardation band was obtained between the FPG protein and double-stranded DNA containing an intact apurinic site or single-stranded DNA containing either an intact or a reduced apurinic site.
大肠杆菌甲酰胺嘧啶 - DNA糖基化酶（FPG蛋白）是一种分子量为30.2 kDa的单体DNA修复酶，已被大量纯化至同质状态。FPG蛋白可从DNA中切除咪唑环打开的嘌呤和8 - 羟基鸟嘌呤残基。除了DNA糖基化酶活性外，FPG蛋白还具有一种抗EDTA活性，可在无嘌呤/无嘧啶位点（AP位点）切割DNA。相比之下，含有化学还原AP位点的DNA不会被FPG蛋白切割。然而，在存在一种纯化的合成24碱基对双链寡核苷酸的情况下，FPG蛋白的DNA糖基化酶活性受到强烈抑制，该寡核苷酸含有一个通过硼氢化钠化学转化为环打开的脱氧核糖衍生物的单个无嘌呤位点。通过在非变性聚丙烯酰胺凝胶中进行电泳研究了FPG蛋白与这种合成修饰DNA形成复合物的能力。FPG蛋白特异性结合含有先前在硼氢化钠存在下还原的无嘌呤位点的双链寡核苷酸。在非变性聚丙烯酰胺凝胶电泳上，该复合物被鉴定为单一的滞后条带。复合物的形成是可逆的，测定的表观解离常数KDapp为2.6×10^(-10) M。相比之下，在FPG蛋白与含有完整无嘌呤位点的双链DNA或含有完整或还原无嘌呤位点的单链DNA之间未获得这样的滞后条带。
