Poly(ADP-ribose) polymerase stimulates DNA polymerase α by physical association

The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP-ribose) polymerase on the activity of DNA polymerase α was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 μg/ml) stimulated the activity of immunoaffinitypurified calf or human DNA polymerase α by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase β, DNA polymerase γ, and primase, indicating that its effect is specific for DNA polymerase α. Apparently, poly(ADP-ribosyl)ation of DNA polymerase α was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP-ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the reaction in competition with template DNA, while, at higher DNA doses, it greatly stimulated the reaction by increasing the F_max of the reaction. By the automodification of poly(ADP-ribose) polymerase, however, both the inhibition at low DNA concentration and the stimulation at high DNA doses were largely lost. Furthermore, stimulation by poly(ADP-ribose) polymerase could not be attributed to its DNA-binding function alone since its fragment, containing only the DNA-binding domain, could not exert full stimulatory effect on DNA polymerase, as of the intact enzyme. Poly(ADP-ribose) polymerase is coimmunoprecipitated with DNA polymerase α, using anti- DNA polymerase α antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase α. In a crude extract of calf thymus, a part of poly(ADP-ribose) polymerase activity existed in a 400-kDa, as well as, a larger 700-kDa complex containing DNA polymerase α, suggesting the existence in vivo of a complex of these two enzymes.