The ability of elevated levels of the AP endonuclease Nfo to increase the wet heat resistance of nfo exoAα−β− spores supports previous suggestions that AP sites are major damaging lesions generated in DNA by wet heat treatment of α−β− spores, and further that AP endonucleases may be important in repairing this damage (Salas-Pacheco et al., 2005). In contrast, overexpression of Nfo in wild-type spores (strain PERM869)
had no effect on these spores’ wet heat resistance (Fig. 2c). Although the nfo exoAα−β− spores with overexpressed Nfo were resistant to wet heat, extended PLX4032 nmr wet heat treatment did result in spore killing (Fig. 2b). This killing is most likely due to damage to some essential protein(s) (Coleman et al., 2007), as there was no increase in auxotrophic and asporogenous mutants among the survivors of extended wet heat treatment of the spores with high Nfo levels (Table 2). In contrast, selleck chemicals llc wet heat treatment of nfo exoAα−β− spores generated a high level of mutants in survivors (Table
2). Nfo overexpression also increased the dry heat resistance of exoA nfoα−β− spores (Fig. 2d). While ∼95% dry spores were killed in 7 min at 90 °C, there was essentially no killing of the exoA nfoα−β− spores with overexpressed Nfo under these conditions. In addition, ∼99% of dry wild-type spores were killed after 120 min at 120 °C, while <10% of dry nfo exoAα−β− spores with overexpressed Nfo were killed under these same conditions (Fig. 2e). Moreover, as shown in Fig. 2f, Nfo overexpression also caused a slight, but significant,
increase in the dry heat resistance of wild-type spores. The increased dry heat resistance of exoA nfoα−β− and wild-type spores with elevated Nfo levels is consistent with dry heat killing of both α−β− and wild-type spores by DNA damage, but more importantly, is consistent with much of this damage being AP lesions. However, the much higher dry heat resistance of exoA nfo PsspB-nfoα−β− spores than wild-type spores with high Nfo levels suggests that dry heat generates DNA damage in addition to AP sites Terminal deoxynucleotidyl transferase in wild-type spores (see Discussion). To investigate whether overexpression of nfo would increase the resistance of nfo exoAα−β− spores to other DNA-damaging treatments, we determined the resistance of spores of various strains to UV-C radiation, a treatment that kills spores almost exclusively by generating photoproducts in DNA (Setlow, 1987, 2006). As expected (Salas-Pacheco et al., 2005), the nfo exoAα−β− spores (and also α−β− spores; Mason & Setlow, 1987) were much more sensitive to UV-C radiation (LD90=30±5 J m−2) than wild-type spores (LD90=274±8 J m−2) (Fig. 3). However, Nfo overexpression did not increase the UV-C resistance of the nfo exoAα−β− spores because they showed an LD90 value of 28±6 J m−2 (Fig. 3).