Naumenko Konstantin N, Berezhnev Egor A, Kurgina Tatyana A, Sukhanova Maria V, Lavrik Olga I
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
Biochemistry (Mosc). 2024 Dec;89(12):2143-2154. doi: 10.1134/S0006297924120046.
Taking into account involvement of the RNA-binding proteins in regulation of activity of poly(ADP-ribose) polymerase 1 (PARP1), a key factor of DNA repair, the effect of the intrinsically disordered protein Sam68 (Src-associated substrate during mitosis of 68 kDa) on catalytic activity of this enzyme was studied. Plasmid containing coding sequence of the Sam68 protein was obtained. Using the obtained construct, conditions for the Sam68 expression in cells were optimized and procedure for protein purification was developed. It was found that Sam68 is able to regulate catalytic activity of PARP1, stimulating auto-poly(ADP-ribosyl)ation of PARP1, interacting with the damaged DNA and purified poly(ADP-ribose) (PAR). Based on the experimental data, a hypothesis on the mechanism of PARP1 activity stimulation by the Sam68 protein was proposed, which involves formation of a complex of Sam68 with poly(ADP-ribosyl)ated PARP1. Sam68 interacts with PAR, shielding its negative charge, which increases the time of PARP1 in the complex with damaged DNA and the overall yield of PAR synthesized by this enzyme.
考虑到RNA结合蛋白参与了DNA修复的关键因子聚（ADP - 核糖）聚合酶1（PARP1）活性的调节，研究了内在无序蛋白Sam68（有丝分裂期间68 kDa的Src相关底物）对该酶催化活性的影响。获得了含有Sam68蛋白编码序列的质粒。利用所得到的构建体，优化了Sam68在细胞中的表达条件，并开发了蛋白质纯化程序。发现Sam68能够调节PARP1的催化活性，刺激PARP1的自身聚（ADP - 核糖基）化，与受损DNA和纯化的聚（ADP - 核糖）（PAR）相互作用。基于实验数据，提出了关于Sam68蛋白刺激PARP1活性机制的假说，该机制涉及Sam68与聚（ADP - 核糖基）化的PARP1形成复合物。Sam68与PAR相互作用，屏蔽其负电荷，这增加了PARP1与受损DNA形成复合物的时间以及该酶合成PAR的总产量。
