Regulatory regions responsive to oxidative stress in the promoter of the human DNA glycosylase gene NEIL2.

Reactive oxygen species (ROS) generated endogenously or from exogenous sources produce mutagenic DNA lesions. If not repaired, these lesions could lead to genomic instability and, potentially, to cancer development. NEIL2 (EC 4.2.99.18), a mammalian base excision repair (BER) protein and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Perturbation in NEIL2 expression may, therefore, significantly impact BER capacity and promote genomic instability. To characterize the genetic and environmental factors regulating NEIL2 gene expression, we mapped the human NEIL2 transcriptional start site and partially characterized the promoter region of the gene using a luciferase reporter assay. We identified a strong positive regulatory region from nucleotide -206 to +90 and found that expression from this region was contingent on its being isolated from an adjacent strong negative regulatory region located downstream (+49 to +710 bp), suggesting that NEIL2 transcription is influenced by both these regions. We also found that oxidative stress, induced by glucose oxidase treatment, reduced the positive regulatory region expression levels, suggesting that ROS may play a significant role in regulating NEIL2 transcription. In an initial attempt to characterize the underlying mechanisms, we used in silico analysis to identify putative cis-acting binding sites for ROS-responsive transcription factors within this region and then used site-directed mutagenesis to investigate their role. A single-base change in the region encompassing nucleotides -206 to +90 abolished the effect of oxidative stress that was observed in the absence of the mutation. Our study is the first to provide an initial partial characterization of the NEIL2 promoter and opens the door for future research aimed at understanding the role of genetic and environmental factors in regulating NEIL2 expression.

Single nucleotide polymorphisms 5' upstream the coding region of the NEIL2 gene influence gene transcription levels and alter levels of genetic damage.

NEIL2 (EC 4.2.99.18), a mammalian DNA glycosylase and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Because base excision repair (BER) proteins act collectively and in a progressive fashion, their proper balance is essential for optimal repair. Thus, inter-individual variability in transcription levels of NEIL2 may predispose to compromised DNA repair capacity and genomic instability by altering the balance of critical BER proteins. In a study of lymphocytes of 129 healthy subjects, using absolute quantitative reverse transcription PCR, we found that NEIL2 transcription varied significantly (up to 63 fold) and that this variability was influenced by certain single nucleotide polymorphisms (SNPs) located 5' of the start site. Using the mutagen sensitivity assay to characterize the biological significance of these SNPs, we observed a significant increase in mutagen-induced genetic damage associated with two SNPs in the promoter region of the NEIL2 gene. To characterize the functional significance of these SNPs, we engineered luciferase-reporter constructs of the NEIL2 promotor with mutations corresponding to these SNPs. We transfected these constructs into MRC-5 cells and evaluated their impact on NEIL2 expression levels. Our results indicate that NEIL2 expression was significantly reduced by over 50% (P < 0.01) in the presence of two SNPs, ss74800505 and rs8191518, located near the NEIL2 start site, which were in significant linkage disequilibrium (D' = 73%; P < 0.05). This first report on in vivo variability in NEIL2 expression in humans identifies SNPs in the NEIL2 promoter region that have functional effects.

The L84F polymorphism in the O6-Methylguanine-DNA-Methyltransferase (MGMT) gene is associated with increased hypoxanthine phosphoribosyltransferase (HPRT) mutant frequency in lymphocytes of tobacco smokers.

OBJECTIVES: O-methylguanine-DNA-methyltransferase (MGMT) is a crucial DNA repair protein that removes DNA adducts formed by alkylating mutagens. Several coding single nucleotide polymorphisms (cSNPs) in the MGMT gene have been reported. Their biological significance, however, is not known. METHODS: We used a newly modified cloning HPRT mutant lymphocyte assay to test the hypothesis that inheritance of the L84F and I143V coding single nucleotide polymorphism in the MGMT gene is associated with increases in HPRT mutant frequency in lymphocytes of individuals exposed to alkylating agents. In addition, we expanded and sequenced 109 mutant clones to test the hypothesis that the mutation spectrum would shift to a larger percentage of base substitutions and G-->A transition mutations in cells with L84F and I143 V coding single nucleotide polymorphisms. RESULTS: We observed no significant effect for the I143 V coding single nucleotide polymorphism on mutant frequency. In contrast, we observed a significant increase in mutant frequency (P<0.01) in lymphocytes from smokers with the 84F coding single nucleotide polymorphism compared with smokers homozygous for the referent L84 wild-type allele. A multiple regression analysis indicated that the mutant frequency increased significantly as a function of the 84F coding single nucleotide polymorphism and smoking, according to the model; mutant frequency (x10)=0.90+0.618 (84F polymorphism)+0.46 (smoking) with R=0.22. Mutation spectra analysis revealed an apparent increase, which was short of statistical significance (P=0.08), in base substitutions in cells with the 84F polymorphism. CONCLUSIONS: These new data suggest that the 84F coding single nucleotide polymorphism may alter the phenotype of the MGMT protein, resulting in suboptimal repair of O-methylguanine lesions after exposure to alkylating agents.

The L84F and the I143V polymorphisms in the O6-methylguanine-DNA-methyltransferase (MGMT) gene increase human sensitivity to the genotoxic effects of the tobacco-specific nitrosamine carcinogen NNK.

O-Methylguanine-DNA-methyltransferase (MGMT) is a direct-reversal DNA repair protein that removes DNA adducts formed by alkylating mutagens found in tobacco smoke. Several coding single nucleotide polymorphisms (cSNPs) in the MGMT gene have been reported. However, their effect on the levels and types of genetic damage induced by specific environmental carcinogens remains to be fully elucidated. We developed two novel genotyping techniques and used them, in conjunction with the mutagen-sensitivity assay, to test the hypothesis that the L84F and I143V cSNPs in the MGMT gene confer increased sensitivity to genetic damage induced by the alkylating tobacco-specific nitrosamine carcinogen NNK. Lymphocytes from 114 healthy volunteers were exposed in vitro to NNK, and the genotoxic response was assessed by measuring chromosome aberration (CA) frequencies. A significant (P<0.02) increase in NNK-induced CA was observed in cells from individuals with the 84F polymorphism compared to cells from individuals homozygous for the referent L84 allele. A significant positive interaction between this cSNP and smoking, gender and age was observed (P<0.03). In subjects with the variant 143V allele, significantly higher levels of NNK-induced CA were observed in males and in young subjects (<43 years old) compared to subjects homozygous for the referent I143 allele (P<0.02). Individuals who inherited two cSNPs had significantly higher levels of NNK-induced CA compared to individuals with none or with one cSNP (P<0.002). These new data suggest that the 84F and 143V cSNPs may alter the function characteristics of the MGMT protein, resulting in suboptimal repair of genetic damage induced by NNK.