ATP for the DNA ligation step in base excision repair is generated from poly(ADP-ribose)

In mammalian cells, the base excision repair (BER) pathway is the main rout e to counteract the mutagenic effects of DNA lesions. DNA nicks induce, amo ng others, DNA polymerase activities and the synthesis of poly(ADP-ribose). It is shown here that poly(ADP-ribose) serves as an energy source for the final and rate-limiting step of BER, DNA ligation. This conclusion was draw n from experiments in which the fate of [P-32]poly(ADP-ribose) or [P-32]NAD added to HeLa nuclear extracts was systematically followed. ATP was synthe sized from poly(ADP-ribose) in a pathway that strictly depended on nick-ind uced DNA synthesis. NAD was used for the synthesis of poly(ADP-ribose), whi ch, in turn, was converted to ATP by pyrophosphorylytic cleavage utilizing the pyrophosphate generated from dNTPs during DNA synthesis. The adenylyl m oiety was then preferentially used to adenylate DNA ligase III, from which it was transferred to the 5'-phosphoryl end of the nicked DNA. Finally, lig ation to the 3'-OH end resulted in the release of AMP. When using NAD, but not poly(ADP-ribose), in the presence of 3-aminobenzamide, the entire proce ss was blocked, confirming poly(ADP-ribosyl)ation to be the essential initi al step. Thus, poly(ADP-ribose) polymerase-1, DNA polymerase beta, and liga se III interact with x-ray repair cross-complementing protein-1 within the BER complex, which ensures that ATP is generated and specifically used for DNA ligation.