Institute of Chemical Biology and Fundamental Medicine (ICBFM), Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Institute of Chemical Biology and Fundamental Medicine (ICBFM), Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Biochimie. 2020 Jan;168:144-155. doi: 10.1016/j.biochi.2019.10.011. Epub 2019 Oct 24.
Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential multifunctional protein in mammals involved in base excision DNA repair (BER), regulation of gene expression and RNA metabolism. Its major enzymatic function is incision of AP sites. Poly(ADP-ribose) polymerase 1 (PARP1) modifies itself and target proteins with poly(ADP-ribose) (PAR), contributing to regulation of many processes. To understand molecular basis of functional cooperation between APE1 and PARP1 in BER, we examined PAR-binding activity and ADP-ribosylation of human APE1 in comparison with known targets of PARP1, using the full-length, N-terminally truncated and catalytically inactive forms of APE1. The protein binds preferentially large ADP-ribose polymers, being very similar to DNA polymerase β (Polβ) but contrasting with the scaffold XRCC1 protein. The interaction with PAR involves the universally conserved catalytic portion and the eukaryote-specific extension of APE1. The ADP-ribosylation of APE1 depends on the structure of PARP1-activating DNA, contrasting APE1 with Polβ and XRCC1. Relative levels of APE1 modification in the presence of different DNA substrates were found to correlate with affinities of the DNAs for APE1 and substrate activities in the enzymatic incision, suggesting the ADP-ribosylation to occur within the DNA-mediated ternary complex. This conclusion was confirmed by importance of the length of DNA region 3' to the AP site for the modification. Deletion of the N-terminal extension of APE1 produced no significant influence on both the ADP-ribosylation efficiency and hydrolytic stability of the modified protein, suggesting localization of target amino acids in the conserved catalytic portion. The most efficient ADP-ribosylation of the catalytically inactive APE1 mutant was shown to reduce the level of PARP1 automodification, suggesting possible role of APE1 in modulating PARP1 activity. Our data on primary role of DNA in controlling the PARP-catalysed modification provide new insights into mechanisms of protein targeting for ADP-ribosylation.
脱嘌呤/脱嘧啶核酸内切酶 1（APE1）是哺乳动物中一种重要的多功能蛋白，参与碱基切除 DNA 修复（BER）、基因表达调控和 RNA 代谢。其主要酶促功能是切割 AP 位点。聚（ADP-核糖）聚合酶 1（PARP1）通过聚（ADP-核糖）（PAR）修饰自身和靶蛋白，有助于调节许多过程。为了了解 APE1 和 PARP1 在 BER 中功能合作的分子基础，我们使用全长、N 端截短和无催化活性的 APE1 形式，比较了人 APE1 的 PAR 结合活性和 ADP-核糖基化与 PARP1 的已知靶标。该蛋白优先结合大的 ADP-核糖聚合物，与 DNA 聚合酶 β（Polβ）非常相似，但与支架 XRCC1 蛋白形成对比。与 PAR 的相互作用涉及普遍保守的催化部分和 APE1 的真核生物特异性延伸。APE1 的 ADP-核糖基化依赖于 PARP1 激活 DNA 的结构，这与 Polβ 和 XRCC1 形成对比。在存在不同 DNA 底物的情况下，APE1 修饰的相对水平与 DNA 与 APE1 的亲和力以及酶切中的底物活性相关，表明 ADP-核糖基化发生在 DNA 介导的三元复合物内。这一结论通过 AP 位点 3' 端的 DNA 区域长度对修饰的重要性得到证实。APE1 的 N 端延伸缺失对修饰的 ADP-核糖基化效率和修饰蛋白的水解稳定性没有显著影响，这表明靶氨基酸定位于保守的催化部分。对无催化活性的 APE1 突变体进行的最有效 ADP-核糖基化降低了 PARP1 自身修饰的水平，这表明 APE1 可能在调节 PARP1 活性方面发挥作用。我们关于 DNA 控制 PARP 催化修饰的主要作用的数据为蛋白质 ADP-核糖基化靶向机制提供了新的见解。
