YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

Abstract

The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonuclease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here. To this end, a recombinant YqfS protein containing an N-terminal His 6 tag was synthesized in Escherichia coli and purified to homogeneity. An anti-His 6 -YqfS polyclonal antibody exclusively localized YqfS in cell extracts prepared from B. subtilis spores. The His 6 -YqfS protein demonstrated enzymatic properties characteristic of the type IV family of DNA repair enzymes, such as AP-endonucleases and 3-phosphatases. However, the purified protein lacked both 5-phosphatase and exonuclease III activities. YqfS showed not only a high level of amino acid identity with E. coli Nfo but also a high resistance to inactivation by EDTA, in the presence of DNA containing AP sites (AP-DNA). These results suggest that YqfS possesses a trinuclear Zn center in which the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules. Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to bind and scan undamaged DNA as well as to strongly interact with AP-DNA. The ability of yqfS to genetically complement the DNA repair deficiency of an E. coli mutant lacking the major AP-endonucleases Nfo and exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of oxidative promoters and alkylating agents. Thus, we conclude that YqfS of B. subtilis is a spore-specific protein that has structural and enzymatic properties required to participate in the repair of AP sites and 3 blocking groups of DNA generated during both spore dormancy and germination.During unpredicted periods of dormancy Bacillus subtilis spores are constantly exposed to environmental conditions that have the potential to cause several types of DNA damage. Therefore, the existence of spore-specific protecting mechanisms would seem to be fundamental for spore survival. One of the factors intricately involved in protecting spore DNA from several types of damage, such as oxidative stress, UV-C irradiation, and desiccation, is the presence of ␣/␤ type small acid-soluble proteins (reviewed in references 16, 28, and 27). Although ␣/␤ type small acid-soluble proteins protect spore DNA from several stresses, they confer protection neither to base alkylation (29) nor to UV-induced DNA strand break formation (30). Thus, while the physiological state of the B. subtilis spores prevents or dramatically slows DNA damage during the long periods of dormancy, it is clear that spores do accumulate potentially lethal and mutagenic DNA lesions such as the spore photoproduct, strand breaks, cyclobutane pyrimidine dimers, chemically altered bases and apurinic/apyridiminic (AP) sites which could affect transcription and replication processes during germination (16,26,29). To remove these potentially deleterious DNA damages and alteratio...

Intrinsic apurinic/apyrimidinic (AP) endonuclease activity enables Bacillus subtilis DNA polymerase X to recognize, incise, and further repair abasic sites

“…Thus, whereas organisms as E.coli and Saccharomyces cerevisiae express ExoIII and EndoIV, representatives of the structural distinct families Xth and Nfo AP endonucleases (30,31), others as Neisseria meningitidis and human cells contain two AP endonucleases belonging to the Xth-type family (32,33). B. subtilis possesses two known AP endonucleases (34), ExoA (Xth), which is expressed in growing cells and in the forespore compartment of the sporulating cell (34), and Nfo, present only in dormant spores (35). Thus, based on their differential expression pattern, only ExoA would be present during the vegetative growth of cell.…”

DNA Repair and Genome Maintenance in Bacillus subtilis

“…B. subtilis contains three AP endonuclease genes: yqfS, exoA, and yshC (14,193). All three AP endonucleases nick the phosphate sugar backbone 5= of the AP site, in a metal-dependent manner, producing a replication-competent 3=-OH and a 5= deoxyribose phosphate terminus (5=-dRp) (339,378). Differences arise between the three AP endonucleases in terms of their spatial localization, temporal expression, and enzymatic activities.…”

“…Differences arise between the three AP endonucleases in terms of their spatial localization, temporal expression, and enzymatic activities. YqfS is a member of the Nfo family (endonuclease IV) of related AP endonucleases (339). Members of this family possess both AP endonuclease and 3=-phosphatase activities yet lack 3=-5= exonuclease function and 5=-phosphatase activities (90).…”

Compartmentalization of Gene Expression duringBacillus subtilisSpore Formation

“…The germination genes include the gerA operon, involved with germination in response to alanine; the homologous gerB operon, involved with germination in response to a panel of germinants (L-asparagine, glucose, fructose, and potassium ions) (213); and pdaA, which is required for the formation of muramic ␦-lactam, a unique component of spore cortex peptidoglycan (89). The genes for protection from DNA damage include splB, encoding spore-photoproduct lyase, which helps protect spore DNA from UV damage (68,219); yqfS, encoding a type IV apurinic/apyrimidinic endonuclease (253,293); and ssp genes, which encode SASPs, the predominant proteins of the spore core (107). Some of the SASPs bind the DNA in the spore and help protect it from exposure to heat, UV radiation, desiccation, and several other adverse conditions and also provide a source of amino acids upon germination.…”