Error-Prone Processing of Apurinic/Apyrimidinic (AP) Sites by PolX Underlies a Novel Mechanism That Promotes Adaptive Mutagenesis in Bacillus subtilis

Abstract

cIn growing cells, apurinic/apyrimidinic (AP) sites generated spontaneously or resulting from the enzymatic elimination of oxidized bases must be processed by AP endonucleases before they compromise cell integrity. Here, we investigated how AP sites and the processing of these noncoding lesions by the AP endonucleases Nfo, ExoA, and Nth contribute to the production of mutations (hisC952, metB5, and leuC427) in starved cells of the Bacillus subtilis YB955 strain. Interestingly, cells from this strain that were deficient for Nfo, ExoA, and Nth accumulated a greater amount of AP sites in the stationary phase than during exponential growth. Moreover, under growth-limiting conditions, the triple nfo exoA nth knockout strain significantly increased the amounts of adaptive his, met, and leu revertants produced by the B. subtilis YB955 parental strain. Of note, the number of stationary-phase-associated reversions in the his, met, and leu alleles produced by the nfo exoA nth strain was significantly decreased following disruption of polX. In contrast, during growth, the reversion rates in the three alleles tested were significantly increased in cells of the nfo exoA nth knockout strain deficient for polymerase X (PolX). Therefore, we postulate that adaptive mutations in B. subtilis can be generated through a novel mechanism mediated by error-prone processing of AP sites accumulated in the stationary phase by the PolX DNA polymerase.

Role of Bacillus subtilis DNA Glycosylase MutM in Counteracting Oxidatively Induced DNA Damage and in Stationary-Phase-Associated Mutagenesis

“…These results suggest the existence of alternative repair pathways that compensate for the absence of MutM; in agreement with this notion, the genomes of the three microorganisms discussed above contain the gene for Nth, a DNA glycosylase capable of processing 8-oxo-G and AP sites (40)(41)(42). In the case of B. subtilis, it was recently shown that the genetic inactivation of Nth not only increases this bacterium's spontaneous Rif r mutation frequency but also sensitizes it to the ROS promoter agent H 2 O 2 (33).…”

“…Taken together, these results strongly suggest that MutM plays a significant role in preventing the cytotoxic effects of 8-oxo-G and possibly of other related lesions, including the opened ring derivative formamidopyrimidine (FaPy) (29)(30)(31)(32), thus contributing to B. subtilis survival. However, in addition to inducing the formation of oxidized bases, H 2 O 2 and PQ may promote other types of DNA lesions, including 8-OxoG·A mispairs and apurinic/apyrimidinic (AP) sites, as well as single-and/or double-strand DNA breaks (33). Therefore, in addition to MutM, other repair proteins, including MutY, Nth, and the AP-endonucleases Nfo and ExoA, most probably contribute to protecting B. subtilis from the genotoxic effects of H 2 O 2 (33).…”

“…However, in addition to inducing the formation of oxidized bases, H 2 O 2 and PQ may promote other types of DNA lesions, including 8-OxoG·A mispairs and apurinic/apyrimidinic (AP) sites, as well as single-and/or double-strand DNA breaks (33). Therefore, in addition to MutM, other repair proteins, including MutY, Nth, and the AP-endonucleases Nfo and ExoA, most probably contribute to protecting B. subtilis from the genotoxic effects of H 2 O 2 (33).…”

Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation

“…To obtain strains harboring a transcriptional aag-lacZ fusion, the integrative vector pMUTIN4 (28) or a chloramphenicol-resistant variant of this plasmid, pMUTIN4cat (29), was utilized. For recombinant plasmid construction, a 360-bp HindIII/BamHI internal fragment of aag (positions ϩ121 to ϩ 480 relative to the aag start codon) was amplified by PCR using chromosomal DNA of B. subtilis 168 and Vent DNA polymerase (New England BioLabs, Ipswich, MA).…”

Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis

“…Moreover, repair of deaminated cytosine in B. subtilis by the uracil DNA glycosylase Ung and the endonuclease V YwqL occurs in an error-prone manner, promoting adaptive mutagenesis (19). A recent report revealed that the numbers of spontaneously or enzymatically generated apurinic/apyrimidinic (AP) sites increase during the stationary phase in B. subtilis and that such lesions can be processed in an error-prone manner by the low-fidelity (LF) DNA polymerase PolX (20). Other reports have documented the participation of LF DNA polymerases, including YqjH (a member of the Y DNA polymerase family), in generating mutations in nutritionally stressed B. subtilis cells (21).…”