Requirement for human AP endonuclease 1 for repair of 3 '-blocking damage at DNA single-strand breaks induced by reactive oxygen species

The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE1) plays a central role in the DNA base excision repair pathway (BER) in two distinct ways, As an AP endonuclease, it initiates repair of Ar sites in DNA produce d either spontaneously or after removal of uracil and alkylated bases in DN A by monofunctional DNA glycosylases, Alternatively, by acting as a 3'-phos phoesterase, it initiates repair of DNA strand breaks with 3'-blocking dama ge, which are produced either directly by reactive oxygen species (ROS) or indirectly through the AP lyase reaction of damage-specific DNA glycosylase s. The endonuclease activity of APE1, however, is much more efficient than its DNA 3'-phosphoesterase activity. Using whole extracts from human HeLa a nd lymphoblastoid TK6 cells, we have investigated whether these two activit ies differentially affect BER efficiency. The repair of ROS-induced DNA str and breaks was significantly stimulated by supplementing the reaction with purified APE1, This enhancement was linearly dependent on the amount of APE 1 added, while addition of other BER enzymes, such as DNA ligase I and FEN1 , had no effect. Moreover, depletion of endogenous APE1 from the extract si gnificantly reduced the repair activity, suggesting that APE1 is essential for repairing such DNA damage and is limiting in extracts of human cells. I n contrast, when uracil-containing DNA was used as the substrate, the effic iency of repair was not affected by exogenous APE1, presumably because the AP endonuclease activity was not limiting. These results indicate that the cellular level of APE1 may differentially affect repair efficiency for DNA strand breaks but not for uracil and AP sites in DNA.