Jurado J, Saparbaev M, Matray T J, Greenberg M M, Laval J
Groupe Réparation des lésions Radio- et Chimio-Induites, UMR 1772 CNRS, Institut Gustave Roussy, Villejuif, France.
Biochemistry. 1998 May 26;37(21):7757-63. doi: 10.1021/bi972982z.
Various forms of oxidative stress, including gamma-radiolysis and UV irradiation, result in the formation of damaged bases. (5R)-Thymidine C5-hydrate is one of several modified nucleosides produced from thymidine under these conditions. N-(2-Deoxy-beta-D-erythro-pentofuranosyl)-N-3-[(2R)-hydroxyisobutyric acid]urea or alphaRT is the respective fragmentation product formed from (5R)-thymidine C5-hydrate upon hydrolysis. This modified nucleoside has potential mutagenic or lethal properties. No enzymatic activity responsible for the removal of alphaRT has been identified. We report here that when present in DNA, alphaRT is a substrate for two purified enzymes from Escherichia coli involved in the repair of oxidized bases: the Nth and the Fpg proteins. The Fpg protein removes the alphaRT lesion more efficiently than the Nth protein. This is the first example of efficient excision of a ring-opened form of a pyrimidine by the Fpg protein. The high efficacy of the Fpg protein suggests that it is likely to be involved in vivo in the excision of alphaRT. The kinetics of the reaction of the Fpg protein with DNA containing alphaRT suggest substrate inhibition. Duplex oligodeoxynucleotides containing alphaRT positioned opposite T, dG, dC, and dA were cleaved efficiently by both enzymes, although the profiles of activity of the two enzymes were different. The Nth enzyme preferentially excises alphaRT when opposite a dG, followed by alphaRT.dA, alphaRT. T, and alphaRT.dC. For the Fpg protein, the order is alphaRT.dC >/= alphaRT.dG approximately alphaRT.T > alphaRT.dA. Moreover, we show that human cell extract exhibits an activity that excises alphaRT from an oligonucleotide, suggesting that human homologues of the Nth and/or Fpg proteins could be involved in repair of this lesion in human cells.
包括γ-辐射分解和紫外线照射在内的各种形式的氧化应激，都会导致碱基受损。（5R）-胸苷C5-水合物是在这些条件下由胸苷产生的几种修饰核苷之一。N-（2-脱氧-β-D-赤藓糖基戊呋喃糖基）-N-3-[(2R)-羟基异丁酸]脲或αRT是（5R）-胸苷C5-水合物水解后形成的相应裂解产物。这种修饰核苷具有潜在的诱变或致死特性。尚未鉴定出负责去除αRT的酶活性。我们在此报告，当存在于DNA中时，αRT是来自大肠杆菌的两种参与氧化碱基修复的纯化酶的底物：Nth和Fpg蛋白。Fpg蛋白比Nth蛋白更有效地去除αRT损伤。这是Fpg蛋白有效切除嘧啶开环形式的首个例子。Fpg蛋白的高效性表明它可能在体内参与αRT的切除。Fpg蛋白与含αRT的DNA反应的动力学表明存在底物抑制。含有与T、dG、dC和dA相对的αRT的双链寡脱氧核苷酸被这两种酶有效切割，尽管这两种酶的活性谱不同。Nth酶在与dG相对时优先切除αRT，其次是αRT.dA、αRT.T和αRT.dC。对于Fpg蛋白，顺序是αRT.dC≥αRT.dG≈αRT.T＞αRT.dA。此外，我们表明人细胞提取物具有从寡核苷酸中切除αRT的活性，这表明Nth和/或Fpg蛋白的人同源物可能参与人细胞中这种损伤的修复。
