Relative affinities of poly(ADP-ribose) polymerase and DNA-dependent protein kinase for DNA strand interruptions

Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK ) are important nuclear enzymes that cooperate to minimize genomic damage c aused by DNA strand interruptions. DNA strand interruptions trigger the ADP -ribosylation activity and phosphorylation activity of PARP and DNA-PK resp ectively. In order to understand the relationship of PARP and DNA-PK with r espect to DNA binding required for their activation, we analyzed the kineti cs of the reactions and determined the apparent dissociation constants (Kd, pp) Of the enzymes for DNA strand interruptions. PARP has a high binding af finity for blunt ends of DNA (K-d app = 116 pM) and 3' single-base overhang s (K-d (app) = 332 pM) in comparison to long overhangs (K-d (app) = 2.6-5.0 nM). Nicks are good activators of PARP although the affinity of PARP for n icks (K-d app = 467 pM) is 4-fold less than that for blunt ends. The K-d (a pp) Of DNA-PK for 3' single-base overhangs, blunt ends and long overhangs i s 704 pM, 1.3 nM and 1.4-2.2 nM respectively. These results demonstrate tha t (1) PARP, when compared to DNA-PK, has a greater preference for blunt end s and 3' single-base overhangs but a weaker preference for long overhangs, and (2) nicks are effective in attracting and activating PARP. The possible implications of the preferences of PARP and DNA-PK for DNA strand interrup tions in vivo are discussed. (C) 1999 Elsevier Science B.V. All rights rese rved.