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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,¹ Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06032,² Department of Biology, University of Nevada-Las Vegas, 4505 Maryland Parkway, Box 454001, Las Vegas, Nevada 89154-400³

Received 7 October 2007/ Accepted 31 December 2007

Germination and outgrowth are critical steps for returning Bacillus subtilis spores to life. However, oxidative stress due to full hydration of the spore core during germination and activation of 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; the outgrowth of spores lacking both of these enzymes was slowed, and the spores had an elevated mutation frequency, suggesting that these enzymes repair DNA lesions induced by oxidative stress during spore germination and outgrowth. Addition of H₂O₂ also slowed the outgrowth of nfo exoA spores and increased the mutation frequency, and nfo and exoA mutations slowed the outgrowth of spores deficient in either RecA, nucleotide excision repair (NER), or the DNA-protective /β-type small acid-soluble spore proteins (SASP). These results suggest that /β-type SASP protect DNA of germinating spores against damage that can be repaired by Nfo and ExoA, which is generated either spontaneously or promoted by addition of H₂O₂. The contribution of RecA and Nfo/ExoA was similar to but greater than that of NER in repair of DNA damage generated during spore germination and outgrowth. However, nfo and exoA mutations increased the spontaneous mutation frequencies of outgrown spores lacking uvrA or recA to about the same extent, 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 /β-type SASP all contribute to the repair of and/or protection against oxidative damage of DNA in germinating and outgrowing spores.

Published ahead of print on 18 January 2008.