Izumi T, Ishizaki K, Ikenaga M, Yonei S
Laboratory of Radiation Biology, Faculty of Science, Kyoto University, Japan.
J Bacteriol. 1992 Dec;174(23):7711-6. doi: 10.1128/jb.174.23.7711-7716.1992.
A mutant allele of the Escherichia coli nfo gene encoding endonuclease IV, nfo-186, was cloned into plasmid pUC18. When introduced into an E. coli xthA nfo mutant, the gene product of nfo-186 complemented the hypersensitivity of the mutant to methyl methanesulfonate (MMS) but not to hydrogen peroxide (H2O2) and bleomycin. These results suggest that the mutant endonuclease IV has normal activity for repairing DNA damages induced by MMS but not those induced by H2O2 and bleomycin. A missense mutation in the cloned nfo-186 gene, in which the wild-type glycine 149 was replaced by aspartic acid, was detected by DNA sequencing. The wild-type and mutant endonuclease IV were purified to near homogeneity, and their apurinic (AP) endonuclease and 3'-phosphatase activities were determined. No difference was observed in the AP endonuclease activities of the wild-type and mutant proteins. However, 3'-phosphatase activity was dramatically reduced in the mutant protein. From these results, it is concluded that the endonuclease IV186 protein is specifically deficient in the ability to remove 3'-terminus-blocking damage, which is required for DNA repair synthesis, and it is possible that the lethal DNA damage by H2O2 is 3'-blocking damage and not AP-site damage.
编码核酸内切酶IV的大肠杆菌nfo基因的一个突变等位基因nfo - 186被克隆到质粒pUC18中。当将其导入大肠杆菌xthA nfo突变体时，nfo - 186的基因产物弥补了该突变体对甲磺酸甲酯（MMS）的超敏性，但对过氧化氢（H2O2）和博来霉素不具有这种作用。这些结果表明，突变的核酸内切酶IV对于修复由MMS诱导的DNA损伤具有正常活性，但对于由H2O2和博来霉素诱导的损伤则没有。通过DNA测序检测到克隆的nfo - 186基因中存在一个错义突变，其中野生型甘氨酸149被天冬氨酸取代。将野生型和突变型核酸内切酶IV纯化至接近均一状态，并测定它们的脱嘌呤（AP）核酸内切酶和3'-磷酸酶活性。野生型和突变型蛋白的AP核酸内切酶活性未观察到差异。然而，突变型蛋白的3'-磷酸酶活性显著降低。从这些结果可以得出结论，核酸内切酶IV186蛋白在去除DNA修复合成所需的3'-末端阻断损伤的能力方面存在特异性缺陷，并且过氧化氢造成的致死性DNA损伤可能是3'-阻断损伤而非AP位点损伤。
