Molecular Mechanisms of Cellular Responses to Radiation and Reactive Oxygen Species

DNA暴露于电离辐射和化学氧化剂中会形成几种胸腺嘧啶氢过氧化物。5-氢过氧甲基尿嘧啶自发分解生成5-甲酰基尿嘧啶（5-foU）和5-羟甲基尿嘧啶（5-hmU）。最近的研究表明，5-foU对细菌和哺乳动物细胞具有潜在的诱变作用。尽管5-hmU被认为是一种主要的辐射产物，但其生物学效应仍不确定。最近，我们发现大肠杆菌Nth、Nei和MutM与含有5-foU或5-hmU的双寡核苷酸形成希夫碱中间体，并在病变部位切割链。作用于氧化碱基的大肠杆菌DNA糖基酶的功能同源物已经在人类细胞中被鉴定出来。hOgg1、hMPG和hNTH1分别与大肠杆菌MutM、AlkA和Nth表现出重叠的底物特异性。此外，最近的研究还发现了大肠杆菌Nei、neil1、NEIL2和NEIL3三个人类同源基因的存在。人类hNEIL1识别与大肠杆菌Nei相似的底物，从DNA中去除甲脒嘧啶，从寡核苷酸中去除氧化嘧啶，如胸腺嘧啶乙二醇。我们之前的工作表明，人类细胞至少具有两种DNA糖基酶活性，可以从DNA中去除5-foU，并且hNTH1具有DNA糖基酶/AP裂解酶活性，可以去除5-foU。除了hNTH1外，人类细胞还应具有另一种类型的5-foU DNA糖基酶。在本报告中，我们发现hNEIL1具有5-foU-和5-hmU-DNA糖基酶活性，可以从双寡核苷酸中切除这些病变。纯化后的hNEIL1通过β-和δ-消除反应裂解含5-foU和5- hmu的寡核苷酸。对5-foU和5-hmU的比活性与胸腺嘧啶乙二醇相当。此外，表达hNEIL1 cDNA可以降低大肠杆菌nthnei突变株的自发突变频率和对过氧化氢的敏感性。

Exposure of DNA to ionizing radiation and chemical oxidants leads to the formation of several thymine hydroperoxides. 5-Hydroperoxymethyluracil formed spontaneously decompose to produce 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU). Recent studies revealed that 5-foU is potentially mutagenic in bacteria and mammalian cells. Despite its identification as a major radiation product, the biological effects of 5-hmU remain uncertain. Recently, we found that the E. coli Nth, Nei and MutM form Schiff base intermediates with duplex oligonucleotides containing 5-foU or 5-hmU and cleave the strand at the lesion site. Functional homologs of E. coli DNA glycosylases that act on oxidized bases have been identified in human cells. hOgg1, hMPG and hNTH1 show overlapping substrate specificities with E. coli MutM, AlkA and Nth, respectively. Furthermore, recent works revealed the presence of three human orthologs of E. coli Nei, hNEIL1, NEIL2 and NEIL3. Human hNEIL1 recognizes similar substrates as E. coli Nei, which excises formamidopyrimidines from DNA and oxidized pyrimidines such as thymine glycol from oligonucleotides. Our previous works revealed that human cells possess at least two kinds of DNA glycosylase activity that remove 5-foU from DNA and showed that hNTH1 had a DNA glycosylase/AP lyase activity for 5-foU. Human cells should possess another type of 5-foU DNA glycosylase in addition to hNTH1. In this report we show that hNEIL1 has a 5-foU- and 5-hmU-DNA glycosylase activity that excises these lesions from duplex oligonucleotides. The purified hNEIL1 cleaved the 5-foU- and 5-hmU-containing oligonucleotides viaβ- and δ-elimination reactions. The specific activities for 5-foU and 5-hmU were comparable with that for thymine glycol. In addition, the expression of hNEIL1 cDNA could reduce the frequency of spontaneous mutation and the sensitivity to hydrogen peroxide in E. coli nthnei mutant strain.