Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA; Weill Cornell Medical College, Cornell University, New York, NY 10065, USA; Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA.
DNA Repair (Amst). 2018 Jun-Jul;66-67:1-10. doi: 10.1016/j.dnarep.2018.04.001. Epub 2018 Apr 17.
Posttranslational modifications of DNA repair proteins have been linked to their function. However, it is not clear if posttranslational acetylation affects subcellular localization of these enzymes. Here, we show that the human DNA glycosylase NEIL1, which is involved in repair of both endo- and exogenously generated oxidized bases via the base excision repair (BER) pathway, is acetylated by histone acetyltransferase p300. Acetylation occurs predominantly at Lys residues 296, 297 and 298 located in NEIL1's disordered C-terminal domain. NEIL1 mutant having the substitution of Lys 296-298 with neutral Ala loses nuclear localization, whereas Lys > Arg substitution (in 3KR mutant) at the same sites does not affect NEIL1's nuclear localization or chromatin binding, presumably due to retention of the positive charge. Although non-acetylated NEIL1 can bind to chromatin, acetylated NEIL1 is exclusively chromatin-bound. NEIL1 acetylation while dispensable for its glycosylase activity enhances it due to increased product release. The acetylation-defective 3KR mutant forms less stable complexes with various chromatin proteins, including histone chaperones and BER/single-strand break repair partners, than the wild-type (WT) NEIL1. We also showed that the repair complex with WT NEIL1 has significantly higher BER activity than the 3KR mutant complex. This is consistent with reduced resistance of non-acetylable mutant NEIL1 expressing cells to oxidative stress relative to cells expressing the acetylable WT enzyme. We thus conclude that the major role of acetylable Lys residues in NEIL1 is to stabilize the formation of chromatin-bound repair complexes which protect cells from oxidative stress.
DNA 修复蛋白的翻译后修饰与其功能有关。然而，尚不清楚翻译后乙酰化是否会影响这些酶的亚细胞定位。在这里，我们表明，人类 DNA 糖苷酶 NEIL1 通过碱基切除修复 (BER) 途径参与修复内源性和外源性生成的氧化碱基，可被组蛋白乙酰转移酶 p300 乙酰化。乙酰化主要发生在 NEIL1 无规卷曲 C 末端结构域中的赖氨酸残基 296、297 和 298 上。具有赖氨酸 296-298 中性 Ala 取代的 NEIL1 突变体失去核定位，而在相同位点的 Lys>Arg 取代（在 3KR 突变体中）不会影响 NEIL1 的核定位或染色质结合，可能是由于保留了正电荷。尽管非乙酰化的 NEIL1 可以与染色质结合，但乙酰化的 NEIL1 仅与染色质结合。NEIL1 的乙酰化虽然对其糖苷酶活性不是必需的，但由于产物释放增加，可增强其活性。与野生型 (WT) NEIL1 相比，缺乏乙酰化的 3KR 突变体与各种染色质蛋白（包括组蛋白伴侣和 BER/单链断裂修复伙伴）形成的复合物不太稳定。我们还表明，具有 WT NEIL1 的修复复合物比 3KR 突变体复合物具有更高的 BER 活性。这与表达不可乙酰化突变体 NEIL1 的细胞对氧化应激的抗性低于表达可乙酰化 WT 酶的细胞一致。因此，我们得出结论，NEIL1 中可乙酰化赖氨酸残基的主要作用是稳定形成染色质结合的修复复合物，从而保护细胞免受氧化应激。
