| Abstract: | The major nuclease activity in nuclei of mycelia of Neurospora crassa has been identified as that of endoexonuclease, an enzyme purified and characterized previously from mitochondria and vacuoles which acts endonucleolytically on single-stranded DNA and RNA and possesses highly processive exonuclease activity with double-stranded DNA. Cross-contamination from the other organelles was eliminated as a source of the activity. Endo-exonuclease of nucleoplasm, chromatin, and nuclear matrix showed 80-100% cross-reaction with antisera raised to purified extranuclear endoexonuclease and was also strongly inhibited by 20 microM aurin tricarboxylic acid. In addition, it yielded some of the same-sized polypeptides on activity gel analysis. Nuclei also contained immunochemically cross-reactive trypsin-activable endo-exonuclease activity, a form of enzyme that was shown previously to occur in high amounts in the cytosol and in a tightly bound form associated with the mitochondrial inner membrane. Pretreatment of wild-type mycelia for 1 h with 4-16 micrograms/ml the DNA-damaging agent, 4-nitroquinoline-1-oxide (4-NQO), which caused about 50-80% growth inhibition, resulted in a dose-dependent loss of up to 80% of inactive endo-exonuclease from nuclei. At low doses of 4-NQO, this was accompanied by increases in the level of active enzyme. Nuclei of the DNA repair-deficient uvs-3 mutant were found to contain only 12% of the active enzyme and about 32% of inactive enzyme as that in wild-type nuclei. Mycelial growth of this mutant was 10 times more sensitive to 4-NQO than the wild-type. At a dose which resulted in equivalent growth inhibition, 4-NQO had no effect on the level of active endo-exonuclease in uvs-3 nuclei and caused an increase (over 30%) in the level of inactive enzyme. These data are consistent with a role of endo-exonuclease in the repair of nuclear DNA. |
