Induction of the Human Oxidized Base-specific DNA Glycosylase NEIL1 by Reactive Oxygen Species

Abstract

NEIL1, a mammalian DNA glycosylase and ortholog of Escherichia coli Nei/Fpg, is involved in the repair of oxidatively damaged bases in mammalian cells. Exposure of HCT116 human colon carcinoma cells to reactive oxygen species, generated by glucose oxidase (GO), enhanced the levels of NEIL1 mRNA and polypeptide by 2-4-fold by 6 h after GO treatment. A similar oxidative stress-induced increase in human NEIL1 (hNEIL1) promoter-dependent luciferase expression in HCT116 cells indicates that reactive oxygen species activates NEIL1 transcription. The transcriptional start site of hNEIL1 was mapped, and the upstream promoter sequence was characterized via luciferase reporter assay. Two identical CRE/AP-1-binding sites were identified in the promoter that binds transcription factors c-Jun and CREB/ATF2. This binding was significantly enhanced in extracts of cells treated with GO. Furthermore, a simultaneous increase in the level of phosphorylated c-Jun suggests its involvement in up-regulating the NEIL1 promoter. Oxidative stress-induced activation of NEIL1 appears to be involved in the feedback regulation of cellular repair activity needed to handle an increase in the level of oxidative base damage.ROS, 2 generated endogenously and as a result of environmental insult, induce oxidative DNA damage, which could affect the integrity of cellular genomes. Oxidative DNA damage, including a multitude of damaged bases, abasic (activator protein (AP)) sites, and DNA strand breaks, is believed to be responsible for sporadic mutations leading to cancer and other pathological as well as age-related syndromes (1-4). Most of the oxidative damage in DNA is repaired via the base excision repair pathway, in which oxidized and altered bases are excised by DNA glycosylases in the first step of repair (5). Base excision repair is highly conserved among all organisms ranging from Escherichia coli to humans. In E. coli, three oxidatively damaged base-specific glycosylases, endonuclease III (Nth), formamidopyrimidine-DNA glycosylase (Fpg), and endonuclease VIII (Nei), excise modified DNA bases with overlapping specificity. Based on structural similarity and reaction mechanism, these enzymes are classified in two families with Nth in one and Nei and Fpg in the other (6 -8). In mammalian cells, only two DNA glycosylases, both belonging to the Nth family, were identified earlier for excision of oxidized bases. NTH1 removes mostly oxidized pyrimidines, e.g. thymine glycol, whereas 8-oxoguanine DNA glycosylase (OGG1), functionally similar to Fpg but mechanistically analogous to Nth, is most active with 8-oxoguanine and formamidopyrimidine-guanine substrates (9, 10). Most surprisingly, both NTH1-and OGG1-null mice are viable and show no major phenotype despite the known mutagenic and toxic effects of their substrate lesions (11, 12). These results suggested the presence of additional DNA glycosylases in mammals that could serve as back-up enzymes for NTH1 and OGG1.We and subsequently several other groups identified and characterized two additional h...

The Human Werner Syndrome Protein Stimulates Repair of Oxidative DNA Base Damage by the DNA Glycosylase NEIL1

“…Significant downregulation of NEIL1 resulted in increased sensitization of mouse embryonic fibroblasts to ionizing radiation (52), in comparison to OGG1-and NTH1-null cells (17,53). We also found up-regulation of hNEIL1 by oxidative stress (37), which underscored its role in protecting cells from the genotoxic effects of ROS. A recent report indicates that NEIL1 null (Neil1 Ϫ/Ϫ ) mice accumulate mitochondrial DNA damage and develop dyslipidemia, obesity, diabetes, and fatty liver disease (54).…”

“…The cell extracts (2 mg/ml) were immunoprecipitated with anti-FLAG M2 antibody (Sigma), washed thoroughly with cold TBS (50 mM Tris-Cl, pH 7.5, 150 mM NaCl), and assayed for the presence of WRN in the complex by immunoblotting with antibody specific for the C terminus of WRN (Bethyl Laboratories). Oxidative stress on cells was imposed by GO treatment as described previously (36,37), and co-immunoprecipitation analyses were carried out with mock and GO-treated cells.…”

“…This confirms that the difference in the lesion level did not result from increased ROS production in the knockdown cells. NEIL1-FLAG were mock treated or treated with a subtoxic dose of GO (100 ng/l), which results in ϳ2.5-fold increase in the ROS level (37). Co-immunoprecipitation analyses revealed greater pull down of WRN by NEIL1-FLAG as a result of GO treatment (Fig.…”

Epigenetic screen of human DNA repair genes identifies aberrant promoter methylation of NEIL1 in head and neck squamous cell carcinoma

“…This observation was experimentally confirmed using a collection of HNSCC cell lines. Since NEIL1 has been shown to be activated by acute oxidative stress 37 and downregulated probably by the long-term exposure to reactive oxygen species in HCV-infected liver cells, 38 it is possible that methylation of NEIL1 promoter region controls the gene expression under these conditions. In addition, major risk factors of HNSCC such as the chronic consumption of tobacco smoke and alcohol can induce a prolonged exposure to reactive oxygen species, 39,40 which might cause epigenetic changes, such as methylation, affecting NEIL1 expression either directly or indirectly.…”

Mutator phenotype of mammalian cells due to deficiency of NEIL1 DNA glycosylase, an oxidized base-specific repair enzyme

“…Changes in intracellular ROS level due to glucose oxidase (GO) treatment were determined as described previously [28], [29]. Briefly, V79 cells were treated with increasing concentrations (10,20,40,60, 80 and 100 ng per ml) of GO for 1h in culture medium and excess GO was removed by washing cells in PBS.…”