Differential specificity of human and Escherichia coli endonuclease III and VIII homologues for oxidative base lesions
Differential specificity of human and Escherichia coli endonuclease III and VIII homologues for oxidative base lesions
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DOI：
10.1074/jbc.m400393200
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发表时间：
2004-04-02
影响因子：
4.8
通讯作者：
Ide, H
中科院分区：
文献类型：
--
作者：
Katafuchi, A;Nakano, T;Ide, H
在人类细胞中，氧化嘧啶损伤通过 Endo III (hNTH1) 和 Endo VIII (hNEIL1 和 hNEIL2) 同源物启动的碱基切除修复途径得到修复。在这项研究中，我们使用确定的寡核苷酸底物定量分析和比较了它们对九个氧化碱基损伤和无嘌呤/无嘧啶（AP）位点的活性。 hNTH1和hNEIL1而非hNEIL2切除了胸腺嘧啶二醇的两种立体异构体（5R-Tg和5S-Tg），但它们的异构体特异性明显不同：hNTH1的5R-Tg: 5S-Tg的相对活性为13:1，hNEIL1的相对活性为1.5:1。其大肠杆菌同系物也是如此：Endo III 的 5R-Tg: 5S-Tg 的相对活性为 1: 2.5，Endo VIII 的相对活性为 3.2: 1。在 hNTH1 的其他测试病变中，AP 位点是比尿素、5-羟基尿嘧啶 (hoU) 和鸟嘌呤衍生的甲酰胺嘧啶 (mFapyG) 明显更好的底物，而对于 hNEIL1，这些基础病变和 AP 位点是相当的底物。相比之下，hNEIL2 专门识别 AP 位点，hoU 和 mFapyG 的活性微乎其微。 hNEIL1、hNEIL2 和 Endo VIII 与 oxanine 形成交联，但 hNTH1 和 Endo III 不形成，表明 Endo VIII 同源物的活性位点的折叠保守。 HeLa 和大肠杆菌细胞提取物切除 Tg 异构体的情况与 hNTH1 和 Endo III 的情况非常相似，证实了它们对细胞中 Tg 异构体修复的主要贡献。然而，对细胞活性的详细分析表明，hNEIL1 在人类细胞中 5S-Tg 的修复中具有重要作用。
In human cells, oxidative pyrimidine lesions are restored by the base excision repair pathway initiated by homologues of Endo III (hNTH1) and Endo VIII (hNEIL1 and hNEIL2). In this study we have quantitatively analyzed and compared their activity toward nine oxidative base lesions and an apurinic/apyrimidinic (AP) site using defined oligonucleotide substrates. hNTH1 and hNEIL1 but not hNEIL2 excised the two stereoisomers of thymine glycol (5R-Tg and 5S-Tg), but their isomer specificity was markedly different: the relative activity for 5R-Tg: 5S-Tg was 13: 1 for hNTH1 and 1.5: 1 for hNEIL1. This was also the case for their Escherichia coli homologues: the relative activity for 5R-Tg: 5S-Tg was 1: 2.5 for Endo III and 3.2: 1 for Endo VIII. Among other tested lesions for hNTH1, an AP site was a significantly better substrate than urea, 5-hydroxyuracil (hoU), and guanine-derived formamidopyrimidine (mFapyG), whereas for hNEIL1 these base lesions and an AP site were comparable substrates. In contrast, hNEIL2 recognized an AP site exclusively, and the activity for hoU and mFapyG was marginal. hNEIL1, hNEIL2, and Endo VIII but not hNTH1 and Endo III formed cross-links to oxanine, suggesting conservation of the - fold of the active site of the Endo VIII homologues. The profiles of the excision of the Tg isomers with HeLa and E. coli cell extracts closely resembled those of hNTH1 and Endo III, confirming their major contribution to the repair of Tg isomers in cells. However, detailed analysis of the cellular activity suggests that hNEIL1 has a significant role in the repair of 5S-Tg in human cells.