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

RESEARCH ARTICLE YwqL (EndoV), ExoA and PolA act in a novel alternative excision pathway to repair deaminated DNA bases in Bacillus subtilis Adriana G. Patlán1☯, Vı́ctor M. Ayala-Garcı́a1☯¤, Luz I. Valenzuela-Garcı́a1, Jimena Meneses-Plascencia1, Pedro L. Vargas-Arias1, Marcelo Barraza-Salas2, Peter Setlow3, Luis G. Brieba4, Mario Pedraza-Reyes ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Patlán AG, Ayala-Garcı́a VM, ValenzuelaGarcı́a LI, Meneses-Plascencia J, Vargas-Arias PL, Barraza-Salas M, et al. (2019) YwqL (EndoV), ExoA and PolA act in a novel alternative excision pathway to repair deaminated DNA bases in Bacillus subtilis. PLoS ONE 14(2): e0211653. https://doi.org/ 10.1371/journal.pone.0211653 Editor: Michael R. Volkert, University of Massachusetts Medical School, UNITED STATES Received: November 19, 2018 Accepted: January 17, 2019 Published: February 6, 2019 Copyright: © 2019 Patlán et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported by: 1) MPR, Grant 221231 National Council of Science and Technology (CONACYT), México; 2) MPR, Grant CIIC 188/2018 University of Guanajuato, México; 3) AGP, VMAG, LIVG, PLVA and JMP were supported by scholarships from CONACyT. The funders had no role in study design, data collection 1 Departamento de Biologı́a, Universidad de Guanajuato, Noria Alta, Guanajuato, Guanajuato, México, 2 Facultad de Ciencias Quı́micas, Universidad Juárez del Estado de Durango, Durango, Durango, México, 3 Department of Molecular Biology and Biophysics, UConn Health, Farmington, Connecticut, United States of America, 4 Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte. Carretera Irapuato-León, Irapuato, Guanajuato, México ☯ These authors contributed equally to this work. ¤ Current address: Facultad de Ciencias Quı́micas, Universidad Juárez del Estado de Durango, Durango, Durango, México. * Abstract 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. PLOS ONE | https://doi.org/10.1371/journal.pone.0211653 February 6, 2019 1 / 22 YwqL(EndoV)-dependent repair of deaminated bases and AP-sites in B. subtilis and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction 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 [6]. To counteract the adverse effects of U, bacteria and mammals rely on repair proteins termed uracil DNA glycosylases (Ung) [7], 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]. Some bacteria and archaea also have a repair protein able to recognize and hydrolyze double-stranded DNA containing a wide spectrum of genetic lesions, including uracil, additional deaminated bases, AP sites, mismatches, flap structures and pseudo-Y structures [10–13]. In E. coli, this enzyme, encoded by the nfi gene (EC: 3.1.21.7), has been termed endonuclease V (EndoV) [14,15]. This Mg2+-dependent enzyme catalyzes the incision of the second phosphodiester bond towards the 3’ end of the lesion [13], thus constituting the first step of an Alternative Excision Repair (AER) pathway [6,16]. Fundamental aspects of catalysis, function and structure of EndoV homologs from distinct organisms have been compiled in an excellent recent review [17]. The genome of the Gram-positive bacterium Bacillus subtilis possess a protein termed YwqL whose amino acid sequence shares 51% identity with that of EndoV (Nfi) from E. coli [18]. It has been shown that YwqL (hereafter BsEndoV) plays a more prominent role than Ung in protecting B. subtilis from the cytotoxic and genotoxic effects of spontaneous and induced factors that promote DNA deamination in this bacterium [18]. Additionally, starved, nongrowing B. subtilis cells lacking ung and ywqL (hereafter endoV) increased their mutation frequency in response to accumulation of DNA lesions [19)]. A recent report revealed that in addition to Ung and BsEndoV, B. subtilis employs Aag, an alkyl-adenine DNA-glycosylase, to help counteract the noxious effects of base deamination [20]. The downstream steps completing the hydrolytic event catalyzed by EndoV on different DNA substrates are currently a matter of investigation. However, the proofreading and polymerase activities of DNA polymerase I (Pol I) are involved in this pathway in E. coli [6, 21, 22]. This proposed mechanism postulates that Pol I recognize (...truncated)