Role of the Nfo and ExoA Apurinic/Apyrimidinic Endonucleases in Repair of DNA Damage during Outgrowth of Bacillus subtilis Spores

Institute for Research in Experimental Biology, Faculty of Chemistry, University of Guanajuato, P.O. Box 187, Guanajuato, Gto. 36050 MEXICO ; Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06032 USA; Department of Biology, University of Nevada-Las Vegas, 4505 Maryland Parkway, Box 454001, Las Vegas, NV 89154-400 USA

^* To whom correspondence should be addressed. Email: pedrama{at}quijote.ugto.mx.

	Abstract

Germination and outgrowth are critical steps for returning Bacillus subtilis spores to life. However, oxidative stress due to full hydration of the spore core in germination and activation of the metabolism in spore outgrowth may generate oxidative DNA damage that in many species is processed by apurinic/apyrimidinic (AP) endonucleases. B. subtilis spores possess two AP endonucleases, Nfo and ExoA, and spores lacking both of these enzymes were slowed in outgrowth and had an elevated mutation frequency suggesting that these enzymes repair DNA lesions induced by oxidative stress during spore germination/outgrowth. H₂O₂ addition also slowed the outgrowth of nfo exoA spores and increased their mutation frequency, and nfo and exoA mutations slowed outgrowth of spores deficient in either RecA, nucleotide excision repair (NER) or the DNA protective /-type small, acid-soluble spore proteins (SASP). These results suggest that /-SASP protect germinating spore DNA against damage repairable by Nfo and ExoA, damage generated either spontaneously or promoted by H₂O₂ addition. RecA and Nfo/ExoA showed a similar but higher contribution than NER in repairing DNA damage generated during spore germination/outgrowth. However, nfo and exoA mutations increased spontaneous mutation frequencies of outgrown spores lacking uvrA or recA to about the same level, suggesting that DNA lesions generated during spore germination and outgrowth are processed by Nfo/ExoA in combination with NER and/or RecA. These results suggest that Nfo/ExoA, RecA, the NER system and /-type SASP all contribute to the repair of and/or protection against oxidative damage in germinating/outgrowing spore DNA.