Hazra Tapas K, Izumi Tadahide, Boldogh Istvan, Imhoff Barry, Kow Yoke W, Jaruga Pawel, Dizdaroglu Miral, Mitra Sankar
Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555, USA.
Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):3523-8. doi: 10.1073/pnas.062053799.
8-oxoguanine (8-oxoG), ring-opened purines (formamidopyrimidines or Fapys), and other oxidized DNA base lesions generated by reactive oxygen species are often mutagenic and toxic, and have been implicated in the etiology of many diseases, including cancer, and in aging. Repair of these lesions in all organisms occurs primarily via the DNA base excision repair pathway, initiated with their excision by DNA glycosylase/AP lyases, which are of two classes. One class utilizes an internal Lys residue as the active site nucleophile, and includes Escherichia coli Nth and both known mammalian DNA glycosylase/AP lyases, namely, OGG1 and NTH1. E. coli MutM and its paralog Nei, which comprise the second class, use N-terminal Pro as the active site. Here, we report the presence of two human orthologs of E. coli mutM nei genes in the human genome database, and characterize one of their products. Based on the substrate preference, we have named it NEH1 (Nei homolog). The 44-kDa, wild-type recombinant NEH1, purified to homogeneity from E. coli, excises Fapys from damaged DNA, and oxidized pyrimidines and 8-oxoG from oligodeoxynucleotides. Inactivation of the enzyme because of either deletion of N-terminal Pro or Histag fusion at the N terminus supports the role of N-terminal Pro as its active site. The tissue-specific levels of NEH1 and OGG1 mRNAs are distinct, and S phase-specific increase in NEH1 at both RNA and protein levels suggests that NEH1 is involved in replication-associated repair of oxidized bases.
8-氧代鸟嘌呤（8-oxoG）、开环嘌呤（甲酰胺嘧啶或Fapys）以及由活性氧产生的其他氧化型DNA碱基损伤通常具有致突变性和毒性，并与包括癌症在内的许多疾病的病因以及衰老有关。所有生物体中这些损伤的修复主要通过DNA碱基切除修复途径进行，该途径始于DNA糖基化酶/AP裂解酶对它们的切除，这些酶分为两类。一类利用内部赖氨酸残基作为活性位点亲核试剂，包括大肠杆菌Nth以及两种已知的哺乳动物DNA糖基化酶/AP裂解酶，即OGG1和NTH1。大肠杆菌MutM及其旁系同源物Nei构成第二类，它们使用N端脯氨酸作为活性位点。在这里，我们报告在人类基因组数据库中存在大肠杆菌mutM nei基因的两个人类直系同源物，并对其中一个产物进行了表征。根据底物偏好，我们将其命名为NEH1（Nei同源物）。从大肠杆菌中纯化至同质的44 kDa野生型重组NEH1可从受损DNA中切除Fapys，并从寡脱氧核苷酸中切除氧化嘧啶和8-氧代鸟嘌呤。由于N端脯氨酸缺失或N端His标签融合导致酶失活，这支持了N端脯氨酸作为其活性位点的作用。NEH1和OGG1 mRNA的组织特异性水平不同，并且在RNA和蛋白质水平上NEH1在S期特异性增加，这表明NEH1参与氧化碱基的复制相关修复。
