Department of Chemistry, University of California, Davis, Davis, California 95616, United States.
Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08854, United States.
J Am Chem Soc. 2022 Aug 17;144(32):14578-14589. doi: 10.1021/jacs.2c03625. Epub 2022 Aug 2.
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.
A-to-I RNA 编辑在人类细胞中广泛存在，但在神经系统外的蛋白质编码区却很少见。一种不寻常的被腺苷脱氨酶 ADAR1 重新编码的靶标是碱基切除 DNA 修复酶 NEIL1 的前体 mRNA，这导致了在损伤识别环中赖氨酸（K）到精氨酸（R）的转换，并改变了底物特异性。通过未编辑（UE，K242）与编辑（Ed，R242）NEIL1 对一系列氧化修饰的 DNA 碱基的碱基去除差异进行了评估，以深入了解影响同种型特异性修复的损伤碱基的化学和结构特征。我们发现，UE NEIL1 对氧化嘧啶的去除活性高于 Ed NEIL1，如胸腺嘧啶二醇、尿嘧啶二醇、5-羟基尿嘧啶和 5-羟甲基尿嘧啶。气相计算表明，相对速率与更稳定的内酰胺互变异构体以及碱基损伤的 N3 的质子亲和力有关。这些趋势支持在 NEIL1 对这些嘧啶碱基损伤的切除催化中，互变异构化和 N3 质子化的贡献。结构相似但不同的底物损伤，5-羟甲基胞嘧啶和胍基乙内酰脲，更有效地被 Ed NEIL1 同种型去除，这与互变异构化、质子亲和力和不稳定性的固有差异一致。我们还观察到具有特定损伤和 NEIL1 同种型组合的双相动力学曲线和不完全碱基去除，提示存在多种损伤结合模式。NEIL1 同种型活性的复杂性意味着 NEIL1 在确保准确修复和作为表观遗传调节剂方面具有多种作用。
