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

Aditi Das,Tapas K Hazra,Istvan Boldogh,Sankar Mitra,Kishor K Bhakat

doi:10.1074/jbc.m505526200

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.

Highlights

ROS,2 generated endogenously and as a result of environmental insult, induce oxidative DNA damage, which could affect the integrity of cellular genomes
NTH1 removes mostly oxidized pyrimidines, e.g. thymine glycol, whereas 8-oxoguanine DNA glycosylase (OGG1), functionally similar to formamidopyrimidine-DNA glycosylase (Fpg) but mechanistically analogous to Nth, is most active with 8-oxoguanine and formamidopyrimidine-guanine substrates [9, 10]. 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
This novel substrate structure preference of NEILs raises the possibility of their specialized in vivo functions for repair of oxidized bases in transient bubbles formed during replication and/or transcription. These results support the scenario that OGG1 and NTH1 are involved primarily in repair of oxidative damage formed in inactive sequences constituting the bulk of the genome

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Chemicals—The human colorectal carcinoma line HCT116 (with wild type p53), a gift of B. To test for basal promoter activity of the upstream sequences, a NEIL1 promoter luciferase reporter construct p(Ϫ2100/ϩ40)NEIL1 luc, containing NEIL1 genomic sequence Ϫ2100 to ϩ40 bp, was used for transient expression in transfected HCT116 cells, as described under “Experimental Procedures.”. The promoter-reporter construct p(Ϫ50) NEIL luc, containing the minimal promoter sequence, showed no up-regulation in luciferase activity under these conditions These results indicate that the cis-acting elements responsible for oxidative stress-induced NEIL1 promoter activity are located 60 –1200 bp 5Ј to the transcriptional start site. Oxidative Stress Activates the NEIL1 Promoter via a Pair of Proximal and Distal CRE/AP-1 Sequences—Examination of the sequence within the Ϫ60- to Ϫ1200-bp region of the NEIL1 promoter revealed two copies of putative elements at positions Ϫ61 and Ϫ1018, both with sequence similarity to the palindromic consensus CRE (TGACGTCA) and AP-1 (TGACTCA) To test whether these CRE-like sites are required for oxidative stress-induced expression of NEIL1, we mutated these sites either individually or simultaneously and assayed the promoter activity, following GO treatment (Fig. 6B). Treatment of HCT116 with GO resulted in a significant increase in the binding of the nuclear extract to TABLE ONE

Primer sequence

Findings

DISCUSSION