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 Fmax 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.
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 05-01-1993|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology