Abstract
Oxidative stress constantly threatens the integrity of cellular DNA under physiological conditions. Flap endonuclease 1 (Fen1) is believed to play a critical role in the repair of oxidized DNA lesions by serving as an integral part of the long-patch (LP)-base excision repair (BER) pathway. The degree to which LP-BER is involved in BER in higher eukaryotes, however, is not clearly understood. Using Fen1-disrupted DT40 cells, we investigated the impact of Fen1 on BER and on DNA replication and cell death under oxidative stress. We found that nuclear extracts from cells deficient in Fen1 had a significant reduction in their LP-BER capacity, and supplementing the extracts with Fen1 later restored LP-BER. Exposure of Fen1-deficient cells to hydrogen peroxide (H2O2) resulted in caspase-dependent cell death within 3 hours. In addition, Fen1-deficient cells had an increase in the amount of stalled replication forks after exposure to? H2O2 as detected by visualization of replication units in combed DNA fibers before and after treatment. Overall, these results strongly suggest that Fen1 has a critical role in the LP-BER process in living higher eukaryotes and imply that Fen1 works as an anti-apoptotic factor during DNA replication possibly by avoiding replication stalled under oxidative stress.
