Human Polymorphic Variants of the NEIL1 DNA Glycosylase

Abstract

In mammalian cells, the repair of DNA bases that have been damaged by reactive oxygen species is primarily initiated by a series of DNA glycosylases that include OGG1, NTH1, NEIL1, and NEIL2. To explore the functional significance of NEIL1, we recently reported that neil1 knock-out and heterozygotic mice develop the majority of symptoms of metabolic syndrome (Vartanian, V., Lowell, B., Minko, I. G., Wood, T. G., Ceci, J. D., George, S., Ballinger, S. W., Corless, C. L., McCullough, A. K., and Lloyd, R. S. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1864 -1869). To determine whether this phenotype could be causally related to human disease susceptibility, we have characterized four polymorphic variants of human NEIL1. Although three of the variants (S82C, G83D, and D252N) retained near wild type levels of nicking activity on abasic (AP) site-containing DNA, G83D did not catalyze the wild type ␤,␦-elimination reaction but primarily yielded the ␤-elimination product. The AP nicking activity of the C136R variant was significantly reduced. Glycosylase nicking activities were measured on both thymine glycol-containing oligonucleotides and ␥-irradiated genomic DNA using gas chromatography/mass spectrometry. Two of the polymorphic variants (S82C and D252N) showed near wild type enzyme specificity and kinetics, whereas G83D was devoid of glycosylase activity. Although insufficient quantities of C136R could be obtained to carry out gas chromatography/mass spectrometry analyses, this variant was also devoid of the ability to incise thymine glycol-containing oligonucleotide, suggesting that it may also be glycosylase-deficient. Extrapolation of these data suggests that individuals who are heterozygous for these inactive variant neil1 alleles may be at increased risk for metabolic syndrome.A major source of DNA lesions in eukaryotic cells is the interaction of reactive oxygen species with DNA constituents.DNA bases are particularly susceptible to reactive oxygen species, with the major DNA damages being, among others, 8-hydroxyguanine (8-OH-Gua), 3 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), and 4,6-diamino-5-formamido pyrimidine (FapyAde) (1, 2). To reverse the potentially deleterious effects of oxidatively induced DNA base lesions, cells primarily utilize the base excision repair pathway to restore the DNA to its original state. This pathway is initiated by lesion-specific DNA glycosylases that hydrolyze the bond attaching the damaged base to the deoxyribose, and many of these enzymes also possess an activity that catalyzes a ␤-or ␤,␦-elimination reaction at the newly formed abasic (AP) site. These incision intermediates are further processed by an AP endonuclease to yield a free 3Ј-OH that serves as a primer for repair synthesis and ligation. To initiate repair of oxidatively induced DNA base lesions, mammalian cells primarily use the products of the ogg1, nth1, neil1, and neil2 genes (reviewed in Ref.2).Human and mouse NEIL1 proteins have been shown to possess a strong substrate preference fo...

Cockayne Syndrome Group B Protein Stimulates Repair of Formamidopyrimidines by NEIL1 DNA Glycosylase

“…FapyGua and FapyAde are substrates for mouse and human NEIL1 (27)(28)(29). Importantly, the results presented here demonstrate a functional and physical interaction between CSB and NEIL1 in vivo and in vitro (Figs.…”

“…NEIL1 excises 8-OH-Gua very inefficiently (27)(28)(29). Here we observed that at least 4-fold more NEIL1 (100 versus 25 fmol) was required to incise a similar fraction of single-lesion DNA substrates containing 8-OH-Gua as FapyGua or 5-OH-Ura (Fig.…”

“…FapyGua and 8-OH-Gua, but not FapyAde, are substrates for OGG1 (44). However, both FapyAde and FapyGua are substrates for NEIL1 (27)(28)(29). This suggests that NEIL1 activity may be compromised in mice lacking CSB and that CSB might cooperate with NEIL1 in recognizing and processing Fapys.…”

“…Formamidopyrimidines (Fapys) 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) are imidazole ring-opened products of purines which share a common intermediate with 8-OHGua and 7,8-dihydro-8-oxoadenine, respectively (11,26). FapyGua and FapyAde are the main substrates of human and mouse NEIL1 (27)(28)(29) and are detected in genomic DNA at a higher level than 8-OH-Gua (30), suggesting that accumulation of these lesions may be of importance in the development of the severe phenotype observed in neil1 Ϫ/Ϫ mice. This study examines the possible role of CSB in repair of FapyGua and FapyAde.…”

NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice

“…If these mouse carcinogenesis studies can be extrapolated to the human populations exposed to various aflatoxins, then deficiencies in the enzymatic activities of NEIL1 may reduce the efficiency of repair of these highly mutagenic adducts. In this regard, others and we have previously carried out biochemical characterization of several NEIL1 polymorphic variants (37,38). When expressed as purified proteins, two of the variants, G83D and C136R, showed little to no DNA glycosylase activity.…”