Activation of topoisomerase I by poly [ADP-ribose] polymerase.
Poly(ADP-ribose) polymerase (PARP I) and Topoisomerase I (Topo I) were reisolated from calf thymus to eliminate cross contamination as tested by immunotransblots. 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 Topo 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 activation 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 and Topo I was demonstrated. The binding of Topo I to circular SV40 DNA, assayed 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 bound to SV40 DNA. These binding experiments provide a molecular basis for the kinetic activation of Topo I by PARP I inasmuch as the increased superhelicity 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 breakage-reunion cycle of Topo I catalysis.
Bauer
P I
PI
Octamer, Inc., San Leandro, CA 94710, USA.
Buki
K G
KG
Comstock
J A
JA
Kun
E
E
D000818:Animals; D002417:Cattle; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004735:Energy Transfer; D004789:Enzyme Activation; D005453:Fluorescence; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification / Q000378:metabolism*
D004270:DNA, Circular; D010446:Peptide Fragments; D009243:NAD; D011065:Poly(ADP-ribose) Polymerases; D004264:DNA Topoisomerases, Type I