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.

Base excision repair of the N-(2-deoxy-d-erythro-pentofuranosyl)-urea lesion by the hNEIL1 glycosylase

“…We observed no differences in rates of excision between urea-containing substrates initially prepared from either oligodeoxynucleotide, as separated by HPLC ( Supplementary Figure S1 in the Supplementary Data). In contrast and consistent with prior reports ( 34 , 43 , 46 , 47 ), excision of Tg by the unedited enzyme was ∼ 50-fold more efficient than by the edited hNEIL1 (Table 2 and Figure 2 ). For the unedited hNEIL1, rates of excision of the urea lesion were ∼30% lower relative to that of Tg.…”

“…Urea lesions are removed by unedited and edited forms of hNEIL1 with comparable efficiencies (Figure 2 and Table 2 ). This contrasts with Tg and several other DNA substrates of hNEIL1 that are excised differentially by unedited vs. edited forms of the glycosylase ( 34 , 37 , 43 , 44 , 46 , 47 , 58 ). The rate of excision of urea lesions by the unedited hNEIL1 (K242) is 30% less than that of Tg, but excision rates by the edited hNEIL1 glycosylase (R242) are greater than that of Tg (Figure 2 and Table 2 ).…”

“…Lotsof et al. ( 46 ) evaluated isoform-specific repair on a series of oxidatively damaged bases. The unedited hNEIL1 glycosylase (K242) exhibited greater activity for oxidized pyrimidine lesions, including Tg, uracil glycol, 5-hydroxyuracil and 5-hydroxymethyluracil, with relative rates of excision by unedited versus edited form being correlated with the ability of these bases to form stable lactim tautomers and the affinity of their N3 atoms to protonation.…”

“…Editing of pre-mRNA encoding h NEIL1 results in an amino acid change at position 242, from lysine in the unedited form of the glycosylase to arginine in the edited form ( 34 , 37 , 44 ). This change has been shown to modulate the rates of base excision on a range of substrates ( 34 , 37 , 43 , 44 , 46 , 47 ), with the unedited form being significantly more efficient in excising Tg lesions than the edited form ( 34 , 43 , 46 , 47 ). hNEIL1 also possesses apurinic/apyrimidinic (AP) lyase activity that catalyzes β-δ elimination ( 48 ), generating a single-nucleotide gap ( 8 , 22–24 , 31 ), with both 5′ and 3′ phosphates ( 8 , 12 , 16 ).…”

“…We envisioned that structural analyses of a complex between the urea lesion and hNEIL1 might not only provide insight into the mechanism by which this glycosylase excises urea lesions, but also advance an understanding of the excision of more abundant Tg and other lesions. An evaluation of the relative activities of edited vs. unedited forms of hNEIL1 against urea lesions was also of interest, as Tg is repaired differentially by the two isoforms of hNEIL1 ( 34 , 43 , 46 , 47 ). Here, utilizing oligodeoxynucleotides containing site-specific urea lesions, we examine hNEIL1-initiated catalysis through a combination of biochemical studies and structural analyses of the urea lesions in DNA and in complex with the active-site mutant CΔ100 P2G hNEIL1 (K242) glycosylase.…”