NEIL1 Recoding due to RNA Editing Impacts Lesion-Specific Recognition and Excision

Abstract

A-to-I RNA editing is widespread in human cells but is uncommon in the coding regions of proteins outside the nervous system. An unusual target for recoding by the adenosine deaminase ADAR1 is the pre-mRNA of the base excision DNA repair enzyme NEIL1 that results in the conversion of a lysine (K) to arginine (R) within the lesion recognition loop and alters substrate specificity. Differences in base removal by unedited (UE, K242) vs edited (Ed, R242) NEIL1 were evaluated using a series of oxidatively modified DNA bases to provide insight into the chemical and structural features of the lesion base that impact isoform-specific repair. We find that UE NEIL1 exhibits higher activity than Ed NEIL1 toward the removal of oxidized pyrimidines, such as thymine glycol, uracil glycol, 5-hydroxyuracil, and 5-hydroxymethyluracil. Gas-phase calculations indicate that the relative rates in excision track with the more stable lactim tautomer and the proton affinity of N3 of the base lesion. These trends support the contribution of tautomerization and N3 protonation in NEIL1 excision catalysis of these pyrimidine base lesions. Structurally similar but distinct substrate lesions, 5-hydroxycytosine and guanidinohydantoin, are more efficiently removed by the Ed NEIL1 isoform, consistent with the inherent differences in tautomerization, proton affinities, and lability. We also observed biphasic kinetic profiles and lack of complete base removal with specific combinations of the lesion and NEIL1 isoform, suggestive of multiple lesion binding modes. The complexity of NEIL1 isoform activity implies multiple roles for NEIL1 in safeguarding accurate repair and as an epigenetic regulator.

Figure 1.. Base lesions evaluated for removal by NEIL1 isoforms produced by RNA editing.

(A) Known substrates for edited NEIL1: Gh, guanidinohydantoin; Sp, spiroiminodihydantoin; FapyA, 4,6-diamino-5-formamidopyrimidine; FapyG, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; DHT, dihydrothymine; 5-OHC, 5-hydroxycytosine; 5-OHU, 5-hydroxyuracil; Tg, thymine glycol. (B) Additional lesions tested as potential substrates for edited and unedited NEIL1 in this work: OI, 8-oxoinosine; Ug, uracil glycol; 5-hmU, 5-hydroxymethyluracil; 5-caC, 5-carboxycytosine; 5-hmC, 5-hydroxymethylcytosine; 5-fC, 5-formylcytosine. C) NEIL1 gene encodes for a lysine at position 242 in NEIL1 enzyme. However, when ADAR1 deaminates adenosine 725 to inosine in the NEIL1 pre-mRNA, this editing event results the encoding of an arginine at position 242 of the NEIL1 enzyme. Both isoforms of NEIL1 are active and remove oxidative damage from DNA.

Figure 2.. Lesion specific removal by edited (Ed) and unedited (UE) NEIL1.

(A) Representative storage phosphor autoradiogram of extent of glycosylase activity of Ed and UE NEIL1 (200 nM) with DHT:A, 5-hmU:C, I:C, 5-OHU:C, OI:C-containing 30-bp duplexes (1•2, 20 nM) at 60 minutes at 37 °C. Modified base removal and strand scission on the duplex substrate leads to a shorter product strand that can be detected via denaturing PAGE. (B) Differences in lesion excision rates (k_g) measured under single-turnover conditions with Ed and UE NEIL1 with a variety of lesions (values listed in Table 1). Values above bar represent the ratio of UE/Ed NEIL1 excision for each lesion. (C) Overall extent of base removed (%) by NEIL1 isoforms with various lesions (values listed in Table 1). The lesion containing DNA duplex substrate (20 nM) was incubated with excess Ed or UE NEIL1 (200 nM) at 37 °C in pH 7.6 buffer containing 150 mM NaCl. The maximal base removed (%) was calculated by dividing the concentration of product produced after a 1 hr reaction by the total concentration of the substrate and multiplying by 100. Error bars are the standard deviation for the end point across three trials.

Figure 3.. Lesion specific removal of uracil glycol versus thymine glycol demonstrate that small differences in lesion structure dramatically impact base excision by NEIL1 isoforms.

Removal and affinity of edited (Ed) and unedited (UE) NEIL1 to duplexes (1•2) containing Ug or Tg was evaluated using rapid quench flow methods with Ed (blue) and UE (black) NEIL1 (200nM) with a DNA duplex (20 nM) containing (A) Ug:C (B) Ug:G (C) Tg:C and (D) Tg:G at 37 C with 150 mM NaCl Data was fit to a two-exponential equation (P = A(1 – exp(−k_g’t)) + B(1 – exp(−k_g”t)). Alternative conformations of residues 242 and 244 of NEIL1 isoforms with dihydrouracil (DHU). (E) X-ray structures of UE (K242, PDB ID: 6LWJ) NEIL1 show residue 242 engaged with DHU lesion in an active conformation. (F) X-ray structures of Ed (R242, PDB ID: 6LWK) NEIL1 show residue 242 away from with DHU lesion and Tyr 242 in a proposed quarantine state.

Figure 4.. Edited (Ed) NEIL1 binds to Tg and Ug containing DNA similarly and slightly tighter than the unedited isoform.

Plot of percent bound enzyme, either D56 K54L Ed NEIL1 or D56 K54L UE NEIL1, versus total enzyme concentrations ([Etotal] = ~[Efree]) for 30-nt duplexes (1•2) containing (A) Ug:G or (B) Tg:G measured by electrophoretic mobility shift assays (EMSA). (C) Reports the values from EMSA and shown in plots (A) and (B). Data was obtained at 25 °C and 150 mM NaCl with 10 pM substrate and enzyme concentrations ranging from 1000 to 0.2 nM. Data was fit to the equation: C[E]ⁿ/((K_d)ⁿ + [E]ⁿ), Hill coefficient = 1.

Figure 5:. Structural features of oxidative damage influence base tautomerization and interaction with NEIL1.

(A, B) X-ray structures of Ed (R242, PDB ID: 5ITY) and UE (K242, PDB ID:5ITX) NEIL1 show that Glu3, Glu6, and Arg/Lys242 make key contacts with the Tg base (C) Correlation of ratio of the lesion excision and favored tautomer. Lesions are ordered based on N3 proton affinity (ΔH kcal/mol, blue) The free base is shown as calculations were done on free base, and excision rates were obtained with duplex DNA (Table 1). (D) Schematic proposal of the impact of the preferred base tautomer on the rate of excision and isoform differences excision.