Suppression of the poly(ADP-ribose) polymerase activity by DNA-dependent protein kinase in vitro

It has been suggested that DNA-dependent protein kinase (DNA-PK) is a centr al component of DNA double-strand-break repair. The mechanism of DNA-PK act ion, however, has not been fully understood. Poly(ADP-ribose) polymerase (P ARP) is another nuclear enzyme which has high affinity to DNA ends. In this study, we analysed the interaction between these tao enzymes. First, DNA-P K was found to suppress the PARP activity and alters the pattern of poly(AD P-ribosyl)ation. Although DNA-PK phosphorylates PARP in a DNA-dependent man ner, this modification is unlikely to be responsible for the suppression of PARP activity, since this suppression occurs even in the absence of ATP. C onversely, PARP was found to ADP-ribosylate DNA-PK in vitro. However, the a utophosphorylation activity of DNA-PK was not influenced by this modificati on. In a competitive electrophoretic mobility shift assay, Ku 70/80 complex , the DNA binding component of DNA-PK, was found to have higher affinity to a short fragment of DNA than does PARP. Furthermore, co-immunoprecipitatio n analysis suggested direct or close association between Ku and PARP. Thus, DNA-PK suppresses PARP activity, probably through direct binding and/or se questration of DNA-ends which serve as an important stimulator for both enz ymes.