YwqL (EndoV), ExoA and PolA act in a novel alternative excision pathway to repair deaminated DNA bases in Bacillus subtilis

DNA deamination generates base transitions and apurinic/apyrimidinic (AP)-sites which are potentially genotoxic and cytotoxic. In Bacillus subtilis uracil can be removed from DNA by the uracil DNA-glycosylase through the base excision repair pathway. Genetic evidence suggests that B . subtilis YwqL, a homolog of Endonuclease-V (EndoV), acts on a wider spectrum of deaminated bases but the factors that complete this pathway have remained elusive. Here, we report that a purified His 6 -YwqL (hereafter Bs EndoV) protein had in vitro endonuclease activity against double-stranded DNAs containing a single uracil (U), hypoxanthine (Hx), xanthine (X) or an AP site. Interestingly, while Bs EndoV catalyzed a single strand break at the second phosphodiester bond towards the 3'-end of the U and AP lesions, there was an additional cleavage of the phosphodiester bond preceding the Hx and X lesions. Remarkably, the repair event initiated by Bs EndoV on Hx and X, was completed by a recombinant B . subtilis His 6 -DNA polymerase A ( Bs PolA), but not on Bs EndoV-processed U and AP lesions. For the latter lesions a second excision event performed by a recombinant B . subtilis His 6 -ExoA ( Bs ExoA) was necessary before completion of their repair by Bs PolA. These results suggest the existence of a novel alternative excision repair pathway in B . subtilis that counteracts the genotoxic effects of base deamination. The presence of this novel pathway in vivo in B . subtilis was also supported by analysis of effects of single or multiple deletions of exoA , endoV and polA on spontaneous mutations in growing cells, and the sensitivity of growing wild-type and mutant cells to a DNA deaminating agent.