Functional characterization of single nucleotide polymorphic variants of DNA repair enzyme NEIL1 in South Asian populations.

The base excision repair (BER) pathway is a precise and versatile mechanism of DNA repair that is initiated by DNA glycosylases. Endonuclease VIII-like 1 (NEIL1) is a bifunctional glycosylase/abasic site (AP) lyase that excises a damaged base and subsequently cleaves the phosphodiester backbone. NEIL1 is able to recognize and hydrolyze a broad range of oxidatively-induced base lesions and substituted ring-fragmented guanines, including aflatoxin-induced 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua). Due to NEIL1's protective role against these and other pro-mutagenic lesions, it was hypothesized that naturally occurring single nucleotide polymorphic (SNP) variants of NEIL1 could increase human risk for aflatoxin-induced hepatocellular carcinoma (HCC). Given that populations in South Asia experience high levels of dietary aflatoxin exposures and hepatitis B viral infections that induce oxidative stress, investigations on SNP variants of NEIL1 that occur in this region may have clinical implications. In this study, the most common South Asian variants of NEIL1 were expressed, purified, and functionally characterized. All tested variants exhibited activities and substrate specificities similar to wild type (wt)-NEIL1 on high-molecular weight DNA containing an array of oxidatively-induced base lesions. On short oligodeoxynucleotides (17-mers) containing either a site-specific apurinic/apyrimidinic (AP) site, thymine glycol (ThyGly), or AFB1-FapyGua, P206L-NEIL1 was catalytically comparable to wt-NEIL1, while the activities of NEIL1 variants Q67K and T278I on these substrates were ≈2-fold reduced. Variant T103A had a greatly diminished ability to bind to 17-mer DNAs, limiting the subsequent glycosylase and lyase reactions. Consistent with this observation, the rate of excision by T103A on 17-mer oligodeoxynucleotides containing ThyGly or AFB1-FapyGua could not be measured. However, the ability of T103A to excise ThyGly was improved on longer oligodeoxynucleotides (51-mers), with ≈7-fold reduced activity compared to wt-NEIL1. Our studies suggest that NEIL1 variant T103A may present a pathogenic phenotype that is limited in damage recognition, potentially increasing human risk for HCC.

Functional analyses of single nucleotide polymorphic variants of the DNA glycosylase NEIL1 in sub-Saharan African populations.

Nei-like glycosylase 1 (NEIL1) is a DNA repair enzyme that initiates the base excision repair (BER) pathway to cleanse the human genome of damage. The substrate specificity of NEIL1 includes several common base modifications formed under oxidative stress conditions, as well as the imidazole ring open adducts that are induced by alkylating agents following initial modification at N7 guanine. An example of the latter is the persistent and mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) adduct, resulting from the alkylating agent aflatoxin B1 (AFB1) exo-8-9-epoxide. Naturally occurring single nucleotide polymorphic (SNP) variants of NEIL1 are hypothesized to be associated with an increased risk for development of early-onset hepatocellular carcinoma (HCC), especially in environments with high exposures to aflatoxins and chronic inflammation from viral infections and alcohol consumption. Given that AFB1 exposures and hepatitis B viral (HBV) infections represent a major problem in the developing countries of sub-Saharan Africa, it is pertinent to study SNP NEIL1 variants that are present in this geographic region. In this investigation, we characterized the three most common NEIL1 variants found in this region: P321A, R323G, and I182M. Biochemical analyses were conducted to determine the proficiencies of these variants in initiating the repair of DNA lesions. Our data show that damage recognition and excision activities of P321A and R323G were near that of wild-type (WT) NEIL1 for both thymine glycol (ThyGly) and AFB1-FapyGua. The substrate specificities of these variants with respect to various oxidatively-induced base lesions were also similar to that of WT. In contrast, the I182M variant was unstable, such that it precipitated under a variety of conditions and underwent rapid inactivation at a biologically relevant temperature, with partial stabilization being observed in the presence of undamaged DNA. This study provides insight regarding the potential increased risk for early-onset HCC in human populations carrying the NEIL1 I182M variant.