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Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid change of lysine 242 to arginine. Previous investigations of the catalytic efficiencies of the two forms of the enzyme revealed differential release of thymine glycol (ThyGly) from synthetic oligodeoxynucleotides, with the unedited form, NEIL1 K242 being ≈30-fold more efficient than the edited NEIL1 K242R. In contrast, when these enzymes were reacted with oligodeoxynucleotides containing guanidinohydantoin or spiroiminohydantoin, the edited K242R form was ≈3-fold more efficient than the unedited NEIL1. However, no prior studies have investigated the efficiencies of these two forms of NEIL1 on either high-molecular weight DNA containing multiple oxidatively-induced base damages, or oligodeoxynucleotides containing a bulky alkylated formamidopyrimidine. To understand the extent of changes in substrate recognition, γ-irradiated calf thymus DNA was treated with either edited or unedited NEIL1 and the released DNA base lesions analyzed by gas chromatography-tandem mass spectrometry. Of all the measured DNA lesions, imidazole ring-opened 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were preferentially released by both NEIL1 enzymes with K242R being ≈1.3 and 1.2-fold more efficient than K242 on excision of FapyAde and FapyGua, respectively. Consistent with the prior literature, large differences (≈7.5 to 12-fold) were measured in the excision of ThyGly from genomic DNA by the unedited versus edited NEIL1. In contrast, the edited NEIL1 was more efficient (≈3 to 5-fold) on release of 5-hydroxycytosine. Excision kinetics on DNA containing a site-specific aflatoxin B1-FapyGua adduct revealed an ≈1.4-fold higher rate by the unedited NEIL1. Molecular modeling provides insight into these differential substrate specificities. The results of this study and in particular, the comparison of substrate specificities of unedited and edited NEIL1 using biologically and clinically important base lesions, are critical for defining its role in preservation of genomic integrity.

Aim: To quantitatively evaluate the inhibition of human DNA repair proteins APE1 and MTH1 by dextran-coated γ-Fe2O3 ultrasmall superparamagnetic iron oxide nanoparticles (dUSPIONs). Materials & methods: Liquid chromatography–tandem mass spectrometry with isotope-dilution was used to measure the expression levels of APE1 and MTH1 in MCL-5 cells exposed to increasing doses of dUSPIONs. The expression levels of APE1 and MTH1 were measured in cytoplasmic and nuclear fractions of cell extracts. Results: APE1 and MTH1 expression was significantly inhibited in both cell fractions at the highest dUSPION dose. The expression of MTH1 was linearly inhibited across the full dUSPION dose range in both fractions. Conclusion: These findings warrant further studies to characterize the capacity of dUSPIONs to inhibit other DNA repair proteins in vitro and in vivo. Inhibitors of DNA repair proteins are increasingly being utilized as potential anticancer agents to supplement traditional chemotherapy and radiation-based approaches. The present study was focused on investigating the use of iron oxide nanoparticles to inhibit the expression of relevant human DNA repair proteins in a cellular model (MCL-5 cells). The authors utilized liquid chromatography–tandem mass spectrometry with isotope dilution to measure the expression levels of two different DNA repair proteins (MTH1 and APE1) in cells after the cells were exposed to increasing levels of the iron oxide nanoparticles. The authors observed significant decreases in DNA repair protein levels that were associated with increasing doses of the iron oxide nanoparticles. The authors' findings warrant more comprehensive studies using other cellular models and suitable animal models. [ABSTRACT FROM AUTHOR]

A study was conducted to investigate the cellular repair of several oxidatively induced DNA lesions in lymphoblasts in women with no detectable breast cancer 1 (BRCA1) deficiency. The results reveal that failure of cells with BRCA1 mutations to repair 8, 5'-cyclopurine-2'-deoxynucleosides indicate the involvement of BRCA1 in nucleotide-excision repair of oxidative DNA damage.

The substrate specificity and excision kinetics of mNEIL1 using high-molecular weight DNA with multiple lesions is investigated. It is concluded that NEIL1 possesses specificity, with similar efficiency of excision, for two purine-derived formamidopyrimidines that are formed in DNA by hydroxyl radical attack or by UV radiation.

Research describes the identification and quantification of 8,5'-cyclo-2'-deoxyguanosine in mammalian DNA by liquid chromatography/isotope dilution- and gas chromatography/isotope dilution mass spectrometry techniques. Data indicate that the levels of (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo are about 2 and 10 lesions per one million DNA molecules, respectively.

BACKGROUND: Exposure to bisphenol A (BPA) has been reported to alter global gene expression, induce epigenetic modifications, and interfere with complex regulatory networks of cells. In addition to these reprogramming events, we have demonstrated that BPA exposure generates reactive oxygen species and promotes cellular survival when co-exposed with the oxidizing agent potassium bromate (KBr[O.sub.3]). OBJECTIVES: We determined the cellular microenvironment changes induced by co-exposure of BPA and KBr[O.sub.3] versus either agent alone. METHODS: Ku70-deficient cells were exposed to 150 µM BPA, 20 mM KBr[O.sub.3], or co-exposed to both agents. Four and 24 hr post-damage initiation by KBr[O.sub.3], with BPA-only samples timed to coincide with these designated time points, we performed whole-genome microarray analysis and evaluated chromatin structure, DNA lesion load, glutathione content, and intracellular pH. RESULTS: We found that 4 hr post-damage initiation, BPA exposure and co-exposure transiently condensed chromatin compared with untreated and KBr[O.sub.3]-only treated cells; the transcription of DNA repair proteins was also reduced. At this time point, BPA exposure and co-exposure also reduced the change in intracellular pH observed after treatment with KBr[O.sub.3] alone. Twenty-four hours post-damage initiation, BPA-exposed cells showed less condensed chromatin than cells treated with KBr[O.sub.3] alone; the intracellular pH of the co-exposed cells was significantly reduced compared with untreated and KBr[O.sub.3]-treated cells; and significant up-regulation of DNA repair proteins was observed after co-exposure. CONCLUSION: These results support the induction of an adaptive response by BPA co-exposure that alters the microcellular environment and modulates DNA repair. Further work is required to determine whether BPA induces similar DNA lesions in vivo at environmentally relevant doses; however, in the Ku70-deficient mouse embryonic fibroblasts, exposure to a high dose of BPA was associated with changes in the cellular microenvironment that may promote survival. http://dx.doi.org/10.1289/EHP237, Introduction World-wide production of bisphenol A (BPA) has increased exponentially as the demand for this chemical in consumer products, from food and beverage containers to epoxies, has grown (Vandenberg et [...]