Date Published: February 6, 2019
Publisher: Public Library of Science
Author(s): Adriana G. Patlán, Víctor M. Ayala-García, Luz I. Valenzuela-García, Jimena Meneses-Plascencia, Pedro L. Vargas-Arias, Marcelo Barraza-Salas, Peter Setlow, Luis G. Brieba, Mario Pedraza-Reyes, Michael R. Volkert.
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 His6-YwqL (hereafter BsEndoV) 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 BsEndoV 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 BsEndoV on Hx and X, was completed by a recombinant B. subtilis His6-DNA polymerase A (BsPolA), but not on BsEndoV-processed U and AP lesions. For the latter lesions a second excision event performed by a recombinant B. subtilis His6-ExoA (BsExoA) was necessary before completion of their repair by BsPolA. 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.
Deamination of DNA bases is one of the most common types of genetic insults in all organisms. Exocyclic amino groups in cytosine, adenine and guanine are particularly vulnerable to spontaneous or chemically induced hydrolytic events [1–5]. Deamination of cytosine, adenine and guanine generates uracil (U), hypoxanthine (Hx) and xanthine (X), respectively, and if not removed from DNA, these lesions promote transition mutations including, CG to TA, AT to GC and GC to AT, respectively . To counteract the adverse effects of U, bacteria and mammals rely on repair proteins termed uracil DNA glycosylases (Ung) , which catalyze the cleavage of the glycosidic bond that connects U with the deoxyribose moiety, generating an apurinic/apyrimidinic (AP) site; this non-coding lesion is further processed by components of the canonical base excision repair pathway (BER) [7,8,9].
In this work, the substrate specificity as well the involvement of BsPolA and BsExoA in the post incision events initiated by BsEndoV on DNA containing deaminated and AP lesions was investigated. We further analyzed the in vivo roles of these enzymes in mutagenesis and resistance to a DNA-deaminating agent. Overall, our results revealed that BsEndoV efficiently nicked DNA containing these lesions and together with BsPolA completed the repair of Hx and X lesions, but an additional enzymatic excision event mediated by BsExoA was necessary to eliminate U and AP lesions through this alternative repair pathway.