RESUMO
Apurinic/apyrimidinic (AP) sites are abundant DNA lesions generated both by spontaneous base loss and as intermediates of base excision DNA repair. In human cells, they are normally repaired by an essential AP endonuclease, APE1, encoded by the APEX1 gene. Other enzymes can cleave AP sites by either hydrolysis or ß-elimination in vitro, but it is not clear whether they provide the second line of defense in living cells. Here, we studied AP site repairs in APEX1 knockout derivatives of HEK293FT cells using a reporter system based on transcriptional mutagenesis in the enhanced green fluorescent protein gene. Despite an apparent lack of AP site-processing activity in vitro, the cells efficiently repaired the tetrahydrofuran AP site analog resistant to ß-elimination. This ability persisted even when the second AP endonuclease homolog, APE2, was also knocked out. Moreover, APEX1 null cells were able to repair uracil, a DNA lesion that is removed via the formation of an AP site. If AP site hydrolysis was chemically blocked, the uracil repair required the presence of NTHL1, an enzyme that catalyzes ß-elimination. Our results suggest that human cells possess at least two back-up AP site repair pathways, one of which is NTHL1-dependent.