Sl. Oei et M. Ziegler, ATP for the DNA ligation step in base excision repair is generated from poly(ADP-ribose), J BIOL CHEM, 275(30), 2000, pp. 23234-23239
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.