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J. Biol. Chem., Vol. 276, Issue 50, 47046-47051, December 14, 2001

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Purification and Characterization of a Bacillus subtilis 168 Nuclease, YokF, Involved in Chromosomal DNA Degradation and Cell Death Caused by Thermal Shock Treatments^*

Jin J. Sakamoto, Miho Sasaki, and Tetsuaki Tsuchido^§¶

From the  Department of Biotechnology, Faculty of Engineering, and ^§ High Technology Research Center, Kansai University, Yamate-cho, Suita 564-8680, Japan

We purified and characterized a 39-kDa Bacillus subtilis 168 nuclease that has been suggested in this laboratory to be involved in chromosomal DNA degradation induced by lethal heat and cold shock treatments in vivo. The nuclease activity was inhibited in vitro by aurintricalboxylic acid but not by Zn²⁺. By the mutant analysis, we identified the 39-kDa nuclease as a product of yokF gene. The yokF gene contained a putative lipoprotein signal peptide motif. After in vivo exposure to lethal heat and cold stresses, the chromosomal DNA fragmentation was reduced in the yokF mutant, which demonstrated about a 2-10-fold higher survival rate than the wild type. The yokF mutant was found to be more sensitive to mitomycin C than the wild type. The transformation efficiency of the yokF mutant was about 10 times higher than that of the wild type. It is suggested that when B. subtilis cells are exposed to a stressful thermal shock resulting in membrane perturbation, YokF nuclease consequently dislocates into the cytoplasm and then attacks DNA.

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