Recognition of DNA adducts by edited and unedited forms of DNA glycosylase NEIL1

Abstract

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.

Keywords: Aflatoxin; Base excision repair; Formamidopyrimidines; Hepatocellular carcinoma; RNA editing; Thymine glycol.

Figure 1.

NEIL1-catalyzed removal of oxidatively-induced DNA base lesions (A) from high-molecular weight genomic DNA. The calf thymus DNA exposed to 5 Gy (B-E) or 20 Gy (F-I) γ-irradiation dose was incubated with no enzyme or in the presence of the unedited or edited NEIL1. The released FapyAde (B & F), FapyGua (C & G), ThyGly (D & H), and 5-OH-Cyt (E & I) were measured by GC-MS/MS using their stable isotope-labeled analogues as internal standards. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. The uncertainties are standard deviations.

Figure 2.

NEIL1-catalyzed excision of AFB₁-FapyGua. The reactions were conducted using 24-mer double-stranded DNA substrate that contained a centrally-located AFB₁-FapyGua (A). Representative gel images for the edited (B) and unedited (C) NEIL1 are shown. The data shown in plot (D) demonstrate the time-dependent product accumulation, with fit of these data that were obtained from three independent experiments (average ± standard deviation) to a single exponential equation (correlation coefficient R > 0.99 for each enzyme).

Figure 3.

Modeling the FapyGua structure in the NEIL1 active site. Structure of ThyGly in the K242 NEIL1 active site (A). Modeled FapyGua in the K242 NEIL1 active site with base in the anti (B) or syn (C) configuration. Selected residues and atoms are labeled and key interactions are indicated with dashed lines.