Date of Award
Doctor of Philosophy
Poly(ADP-ribose) polymerase from calf thymus was purified to near homogeneity. The use of red-agarose resulted in a rapid purification with a high yield. Purified poly(ADP-ribose) polymerase was inhibited by 1,10-phenanthroline, a metal chelating agent, at pH < 8. This inhibition and the inhibition by other chelating agents suggested that this enzyme was a metalloprotein. Control experiments eliminated the possibility that the inhibition was due to the DNA-degrading properties of 1,10-phenanthroline. Atomic absorption spectroscopy showed the presence of one atom of zinc per protein molecule. Dialysis of the enzyme against buffers containing 1,10-phenanthroline resulted in the loss of activity and the coincidental removal of zinc from the enzyme. Initial rate kinetics showed that 1,10-phenanthroline was non-competitive with NAD('+) and competitive with DNA. The binding of DNA to the enzyme was unaffected by the inhibitor. These results suggest a metal-containing site is involved in the interaction of DNA and poly(ADP-ribose) polymerase.;Previous reports have indicated that poly(ADP-ribose) polymerase, besides modifying various chromatin proteins, also modifies itself. The studies reported here indicate that this auto-modification inhibited the enzyme. By observing the binding of poly(ADP-ribose) polymerase to the DNA it was shown that the affinity of modified poly(ADP-ribose) polymerase for DNA was decreased. Mg('2+) and histone H1 appear to activate the polymerase by increasing the affinity of the auto-modified polymerase for DNA, probably by neutralizing the negative charges on poly(ADP-ribose). The coupling of poly(ADP-ribose) glycohydrolase with the polymerase reactivated the polymerase by degrading the poly(ADP-ribose) and restoring the polymerase-DNA complex. These results provided the basis for a shuttle mechanism by which proteins could be moved on and off DNA by the actions of poly(ADP-ribose) polymerase and glycohydrolase.
Zahradka, Peter, "Poly(adp-ribose) Polymerase: Aspects Of Its Interaction With Dna" (1984). Digitized Theses. 1340.