Kurgina T A, Anarbaev R O, Sukhanova M V, Lavrik O I
Institute of Chemical Biology and Fundamental Medicine, Lavrentiev av. 8, 630090 Novosibirsk, Russia; Department of Natural Sciences, Novosibirsk State University, 2 Pirogov Street, 630090 Novosibirsk, Russia.
Institute of Chemical Biology and Fundamental Medicine, Lavrentiev av. 8, 630090 Novosibirsk, Russia.
Anal Biochem. 2018 Mar 15;545:91-97. doi: 10.1016/j.ab.2017.12.033. Epub 2018 Jan 8.
Poly(ADP-ribose) polymerase 1 (PARP1) is a key enzyme that regulates important cellular processes, including DNA repair. PARP1 binds to a DNA damage site and synthesizes poly(ADP-ribose) chains (PARs), which serve as a signal of DNA damage for other DNA repair enzymes. PARP1 is a recognized target for the development of anti-cancer drugs. In this work, a method is developed that makes it possible to investigate the complex formation of PARP1 with DNA as well as its dissociation by detecting the fluorescence anisotropy of this complex during the poly(ADP-ribose) synthesis. The method allows investigation of the inhibition of PARP1 activity in the presence of its inhibitors. In this work, we demonstrated that PARP1 is activated by DNA duplexes containing a damage and a fluorophore at the 3'-end of one of the DNA duplex chains. The effects of the clinical inhibitor olaparib on the activity of PARP1 was studied. It was shown that olaparib has no influence on the binding of PARP1 to the model DNA structures used, but it significantly inhibits the poly(ADP-ribosyl)ation of PARP1. The proposed convenient method for the real-time determination of the PARP1 activity can be used to screen PARP1 inhibitors with the calculation of quantitative inhibition parameters.
聚（ADP - 核糖）聚合酶1（PARP1）是一种关键酶，可调节包括DNA修复在内的重要细胞过程。PARP1与DNA损伤位点结合并合成聚（ADP - 核糖）链（PARs），这些链作为其他DNA修复酶的DNA损伤信号。PARP1是抗癌药物开发的公认靶点。在这项工作中，开发了一种方法，通过在聚（ADP - 核糖）合成过程中检测该复合物的荧光各向异性，能够研究PARP1与DNA的复合物形成及其解离。该方法允许在存在抑制剂的情况下研究PARP1活性的抑制作用。在这项工作中，我们证明PARP1被一种DNA双链体激活，该双链体在其中一条DNA双链链的3'端含有损伤和荧光团。研究了临床抑制剂奥拉帕利对PARP1活性的影响。结果表明，奥拉帕利对PARP1与所用模型DNA结构的结合没有影响，但它显著抑制PARP1的聚（ADP - 核糖基）化。所提出的用于实时测定PARP1活性的便捷方法可用于筛选PARP1抑制剂并计算定量抑制参数。
