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.

中文翻译：

---

通过编辑和未编辑形式的DNA糖基化酶NEIL1识别DNA加合物。

编码人NEIL1的pre-mRNA受到腺苷脱氨酶ADAR1的编辑，该酶将单个腺苷转化为肌苷，这种转化导致赖氨酸242的氨基酸变为精氨酸。先前对这两种酶的催化效率的研究表明，胸腺嘧啶二醇（ThyGly）从合成的寡脱氧核苷酸中释放出来，未经编辑的形式，NEIL1 K242的效率比经编辑的NEIL1 K242R约高30倍。相反，当这些酶与含有胍基乙内酰脲或螺氨基乙内酰脲的寡脱氧核苷酸反应时，经编辑的K242R形式的效率要比未经编辑的NEIL1高约3倍。但是，之前的研究都没有研究这两种形式的NEIL1对包含多个氧化诱导的碱基损伤的高分子量DNA的效率，或含有庞大的烷基化甲酰胺基嘧啶的寡脱氧核苷酸。为了了解底物识别变化的程度，对γ射线照射的小牛胸腺DNA进行了编辑或未编辑的NEIL1处理，并通过气相色谱-串联质谱分析了释放的DNA碱基病变。在所有测得的DNA损伤中，两种NEIL1酶均优先释放咪唑开环的4,6-二氨基-5-甲酰胺基嘧啶（FapyAde）和2,6-二氨基-4-羟基-5-甲酰胺基嘧啶（FapyGua），K242R为切除FapyAde和FapyGua的效率分别比K242高≈1.3和1.2倍。与现有文献一致，未编辑的NEIL1与编辑的NEIL1在从基因组DNA切除ThyGly时测得较大差异（约7.5至12倍）。相比之下，编辑的NEIL1在释放5-羟基胞嘧啶方面更有效（约3至5倍）。含有特定位置的黄曲霉毒素B1-FapyGua加合物的DNA的剪切动力学显示，未经编辑的NEIL1的速率高出约1.4倍。分子建模可以洞悉这些底物差异特异性。这项研究的结果，尤其是使用生物学和临床上重要的基础病变对未编辑和已编辑NEIL1底物特异性的比较，对于定义其在保存基因组完整性中的作用至关重要。