A monofunctional-like mutant of DNA glycosylase NTHL1 changes the dynamics of DNA repair during acute oxidative stress.

Bifunctional DNA glycosylases, which initiate the base excision repair (BER) of oxidized bases, act by first excising the base and then incising the DNA backbone. In vitro, these enzymes are often rate-limited by their apurinic/apyrimidinic (AP)-lyase activity; however, the significance of this step in cells has remained unclear, because AP-endonuclease 1 (APE1) can efficiently bypass this step. To analyze the importance of the AP-lyase activity of NTHL1, we rationally designed and characterized a monofunctional-like NTHL1 mutant with glycosylase activity but profoundly impaired AP-lyase activity using complementary biochemical and microscopy-based assays. Mechanistically, we demonstrated that the monofunctional-like NTHL1 mutant generates abasic sites (AP sites) but, despite lacking effective AP-lyase activity, remains AP site bound. This creates competition with APE1 for engagement of AP sites. Moreover, cells expressing the monofunctional-like NTHL1 mutant accumulated more AP sites, retained higher levels of XRCC1 foci and displayed heightened sensitivity to acute oxidative stress. Live-cell assays further revealed increased NTHL1 accumulation at laser-induced DNA damage sites and increased chromatin bound immobility during oxidative stress, with mobility restored after a repair period. In contrast, a catalytically inactive NTHL1 mutant was recruited less strongly but remained chromatin-bound for a longer time. Thus, in contrast to the monofunctional-like NTHL1 mutant, the bifunctional NTHL1 limits BER intermediate retention and enables timely hand-off to downstream enzyme APE1. Ultimately, disrupting the AP-lyase ability of NTHL1 disrupts BER pathway flux and affects chromatin engagement during oxidative stress.