Identification and characterization of base excision repair enzymes involved in the repair of oxidative DNA damage

在这项研究中，我们对哺乳动物DNA糖基酶进行了鉴定和鉴定，以阐明哺乳动物细胞中DNA氧化损伤的修复机制。本研究的主要结果如下：(1)以含口恶嘌呤的寡核苷酸为探针，采用基于机理的捕获方法，从HeLa细胞提取液中分离出DNA损伤识别蛋白。经SDS-PAGE分析，约有14个蛋白质(28-69 kDa)为捕获产物。对捕获产物的大量指纹分析表明，与组蛋白一起，有几种蛋白质可能参与DNA修复。对其功能的详细分析正在进行中。(2)从大鼠肝脏中分离得到5-甲酰尿嘧啶(5-FU)的DNA糖基酶活性，鉴定为SMUG1。人SMUG1识别尿嘧啶及其C5环上含氧化基团的衍生物，即氟尿嘧啶、5-羟甲基尿嘧啶和5-羟基尿嘧啶。在HeLa细胞中，SMUG1是这些损伤的主要活性成分。因此，SMUG1是参与氧化损伤修复的DNA糖基酶的一个新成员。(3)人糖基酶(hNTH1、hNEIL1和hNEIL2)的损伤特异性已经被表征，并与大肠杆菌的相应酶进行了比较。尽管人和大肠杆菌同源物(Endo III vs.hNTH1，Endo VIII vs.hNEIL1)显示出显著不同的损伤偏好，但hNEIL2对胸腺嘧啶二醇立体异构体和甲酰胺比目标物显示出非常弱的N-糖基酶活性。(4)对E.ColiEndo IV和酵母APN1的修复活性分析表明，它们启动了自由基诱导的DNA损伤的核苷酸切割修复(NIR)。NIR可能构成细胞内碱基切除修复(BER)途径的替代或备用修复途径。

In this study, we have performed identification and characterization of mammalian DNA glycosylases to elucidate the repair mechanism of oxidative DNA damage in mammalian cells. The findings of this research are summarized as follows.(1) DNA damage recognition proteins were isolated from HeLa cell extracts by mechanism-based trapping assays using oxanine-containing oligonucleotides as probes. About 14 proteins (28-69 kDa) were identified as trapped products in SDS-PAGE analysis. Mass fingerprinting analysis of trapped products indicates, together with histone, several proteins that might be involved in DNA repair. Detailed analysis of their function is ongoing.(2) DNA glycosylase activity for 5-formyluracil (fU) was isolated from rat liver and identified as SMUG1. Human SMUG1 recognized uracil and its derivatives bearing an oxidized group at the ring C5 position, i.e., fU, 5-hydroxymethyluracil, and 5-hydroxyuracil. SMUG1 accounted for dominant activities for these lesions in HeLa cells. Thus, SMUG1 is a new member of DNA glycosylases involved in the repair of oxidative damage.(3) The damage specificities of human glycosylases (hNTH1, hNEIL1, and hNEIL2) have been characterized and compared to those of E.coli counterparts. Despite being homologues, human and E.coli homologues (Endo III vs.hNTH1, Endo VIII vs.hNEIL1) exhibit significantly different damage preferences, particularly, for thymine glycol stereoisomers and formamidopymidine, hNEIL2 exhibits only very weak N-glycosylase activity.(4) Analysis of repair activity of E.coli Endo IV and yeast APN1 suggests that they initiate nucleotide incision repair (NIR) for free radical-induced DNA lesions. NIR may constitute an alternative or backup repair pathway for the base excision repair (BER) pathway in cells.