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

• Published in: PloS one Vol. 14; no. 2; p. e0211653
• Main Authors: Patlán, Adriana G, Ayala-García, Víctor M, Valenzuela-García, Luz I, Meneses-Plascencia, Jimena, Vargas-Arias, Pedro L, Barraza-Salas, Marcelo, Setlow, Peter, Brieba, Luis G, Pedraza-Reyes, Mario
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 06.02.2019; Public Library of Science (PLoS)
• Subjects: Antifungal agents; Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacterial genetics; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base excision repair; Biology and Life Sciences; Bonds (Securities); Cloning; Crystal structure; Cytotoxicity; Deamination; Deoxyribonuclease (Pyrimidine Dimer) - genetics; Deoxyribonuclease (Pyrimidine Dimer) - metabolism; Deoxyribonucleic acid; DNA; DNA damage; DNA glycosylases; DNA polymerase; DNA Polymerase I - genetics; DNA Polymerase I - metabolism; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase - metabolism; DNA-directed DNA polymerase; E coli; Endonuclease; Enzymes; Genetic aspects; Genomes; Genotoxicity; Homology; Hypoxanthine; Laboratories; Lesions; Medicine and Health Sciences; Microbiological research; Mutagenesis; Mutation; Nucleases; Physical Sciences; Polymerase; Proteins; Purines; Pyrimidines; Recombinant Proteins; Repair; Research and Analysis Methods; Uracil; Xanthine
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0211653

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.