Hori N, Iwai S, Inoue H, Ohtsuka E
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
J Biol Chem. 1992 Aug 5;267(22):15591-4.
T4 endonuclease V recognizes thymine photodimers in DNA duplexes and, in a two-step reaction, cleaves the glycosyl linkage of the 5'-side thymidine and the phosphodiester linkage. To determine the amino acid residues responsible for binding thymine photodimers, a photoaffinity reagent, 4-(1-azi-2,2,2-trifluoroethyl)-benzoate, was linked to the aminoalkylphosphonate of a thymine photodimer in a 14-mer duplex. The reactive substrate was treated with the enzyme under UV light (365 nm). The nascent enzyme and the modified enzyme were treated with lysyl endopeptidase, and the peptide maps were compared. Three peptides from the C terminus were found to interact with the reactive oligonucleotide to various extents. The three modified peptides were isolated and analyzed by Edman degradation. The amino acid residues Gly-133, Tyr-129, and Thr-89 were partially linked with the reactive substrate and may be involved in the binding of thymine photodimers.
T4 核酸内切酶 V 可识别 DNA 双链中的胸腺嘧啶光二聚体，并通过两步反应切割 5' 侧胸腺嘧啶核苷的糖苷键和磷酸二酯键。为了确定负责结合胸腺嘧啶光二聚体的氨基酸残基，将一种光亲和试剂 4-(1-叠氮基-2,2,2-三氟乙基)-苯甲酸酯与 14 聚体双链体中胸腺嘧啶光二聚体的氨基烷基膦酸酯相连。在紫外光（365 nm）下用该酶处理反应性底物。用赖氨酰内肽酶处理新生酶和修饰酶，并比较肽图谱。发现来自 C 末端的三个肽与反应性寡核苷酸有不同程度的相互作用。分离出这三个修饰肽并通过埃德曼降解法进行分析。氨基酸残基 Gly-133、Tyr-129 和 Thr-89 与反应性底物部分相连，可能参与胸腺嘧啶光二聚体的结合。
