Activation of topoisomerase I by poly [ADP-ribose] polymerase

Poly(ADP-ribose) polymerase (PARP I) and Topoisomerase I(Topo I) were reiso lated from calf thymus to eliminate cross contamination as tested by immuno transblots. The specific activity of Topo I was greatly increased by added PARP I, following saturation kinetics. Recombinant PARP I and isolated PARP I at final purity were indistinguishable in terms of their activation of T opo I. There was a coincidence of experimentally obtained binding constants and computer generated values based on the kinetic model, indicating that the association of PARP I and Topo I is rate limiting in the catalytic acti vation of Topo I by PARP I. Polypeptide domains of PARP I that are required for protein-protein binding and protein-DNA binding also activate Topo I. Fluorescence resonance energy transfer between fluorophor-labeled PARP I an d Topo I was demonstrated. The binding of Topo I to circular SV40 DNA, assa yed either by the formation of a) the sum of non-covalently and covalently attached Topo I to DNA or b) by the covalently bound transient intermediate in the presence of camptothecin, was augmented when PARP I protein was bou nd to SV40 DNA. These binding experiments provide a molecular basis for the kinetic activation of Topo I by PARP I inasmuch as the increased superheli city of SV40 DNA induced by PARP I may facilitate the formation of a more ' tight fisted' Topo I-DNA complex that increases the rate of the DNA breakag e-reunion cycle of Topo I catalysis.