Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, VT 05405-0086, USA.
Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):4925-30. doi: 10.1073/pnas.0908307107. Epub 2010 Feb 25.
To protect cells from oxidative DNA damage and mutagenesis, organisms possess multiple glycosylases to recognize the damaged bases and to initiate the Base Excision Repair pathway. Three DNA glycosylases have been identified in mammals that are homologous to the Escherichia coli Fpg and Nei proteins, Neil1, Neil2, and Neil3. Neil1 and Neil2 in human and mouse have been well characterized while the properties of the Neil3 protein remain to be elucidated. In this study, we report the characterization of Mus musculus (house mouse) Neil3 (MmuNeil3) as an active DNA glycosylase both in vitro and in vivo. In duplex DNA, MmuNeil3 recognizes the oxidized purines, spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino- 5-formamidopyrimidine (FapyA), but not 8-oxo-7,8-dihydroguanine (8-oxoG). Interestingly, MmuNeil3 prefers lesions in single-stranded DNA and in bubble structures. In contrast to other members of the family that use the N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine. We expressed the glycosylase domain of MmuNeil3 (MmuNeil3Delta324) in an Escherichia coli triple mutant lacking Fpg, Nei, and MutY glycosylase activities and showed that MmuNeil3 greatly reduced both the spontaneous mutation frequency and the level of FapyG in the DNA, suggesting that Neil3 plays a role in repairing FapyG in vivo.
为了保护细胞免受氧化 DNA 损伤和诱变，生物体拥有多种糖苷酶来识别受损碱基并启动碱基切除修复途径。在哺乳动物中已经鉴定出三种与大肠杆菌 Fpg 和 Nei 蛋白同源的 DNA 糖苷酶，即 Neil1、Neil2 和 Neil3。人类和小鼠中的 Neil1 和 Neil2 已经得到了很好的表征，而 Neil3 蛋白的性质仍有待阐明。在这项研究中，我们报告了 Mus musculus（家鼠）Neil3（MmuNeil3）作为一种在体外和体内都具有活性的 DNA 糖苷酶的特性。在双链 DNA 中，MmuNeil3 识别氧化嘌呤、螺环亚胺二氢嘧啶（Sp）、胍基二氢嘧啶（Gh）、2,6-二氨基-4-羟基-5-甲酰胺嘧啶（FapyG）和 4,6-二氨基-5-甲酰胺嘧啶（FapyA），但不识别 8-氧代-7,8-二氢鸟嘌呤（8-oxoG）。有趣的是，MmuNeil3 更喜欢单链 DNA 和泡状结构中的损伤。与家族中的其他成员不同，它们使用 N 端脯氨酸作为亲核试剂，MmuNeil3 通过其 N 端缬氨酸形成席夫碱中间体。我们在缺乏 Fpg、Nei 和 MutY 糖苷酶活性的大肠杆菌三重突变体中表达了 MmuNeil3 的糖苷酶结构域（MmuNeil3Delta324），并表明 MmuNeil3 大大降低了 DNA 中的自发突变频率和 FapyG 水平，这表明 Neil3 在体内修复 FapyG 中发挥作用。
