Effects of the RNA-Binding Protein Sam68 on Poly(ADP-Ribose)polymerase 1 Activity.

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.