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

在这项研究中，我们已经进行了鉴定和哺乳动物DNA糖基化酶的特性，以阐明在哺乳动物细胞中的氧化DNA损伤的修复机制。本研究的结果总结如下。(1)DNA损伤识别蛋白的分离，从HeLa细胞提取物的机制为基础的捕获分析使用含？？经SDS-PAGE分析，约有14种蛋白质（28-69 kDa）被鉴定为捕获产物。对捕获产物的大规模指纹分析表明，与组蛋白一起，可能涉及DNA修复的几种蛋白质。目前正在对其职能进行详细分析。(2)从大鼠肝脏中分离5-甲酰尿嘧啶（fU）的DNA糖基化酶活性，并鉴定为SMUG 1。人SMUG 1识别在环C5位置带有氧化基团的尿嘧啶及其衍生物，即，fU、5-羟甲基尿嘧啶和5-羟基尿嘧啶。SMUG 1占主导地位的活动，这些病变在HeLa细胞。因此，SMUG 1是参与氧化损伤修复的DNA糖基化酶的新成员。(3)人糖基化酶（hNTH 1、hNEIL 1和hNEIL 2）的损伤特异性已经被表征并与大肠杆菌对应物的损伤特异性进行比较。尽管是同源物，但人和大肠杆菌同源物（Endo III vs.hNTH1，Endo VIII vs.hNEIL1）表现出显著不同的损伤偏好，特别是对于胸腺嘧啶二醇立体异构体和甲酰胺基嘧啶，hNEIL 2仅表现出非常弱的N-糖基化酶活性。(4)对大肠杆菌Endo IV和酵母APN 1的修复活性的分析表明，它们启动了自由基诱导的DNA损伤的核苷酸切口修复（NIR）。近红外可能构成细胞中碱基切除修复（BER）途径的替代或备份修复途径。

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.