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Mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae is organized to form mitochondrial nucleoids (mt-nucleoids) by association with specific proteins. The sensitivity of DNA-protein complexes to nuclease digestion is a useful means for examining DNA packaging and organization. However, the mt-nucleoids isolated from wild-type cells of S. cerevisiae demonstrated a significant amount of endogenous nuclease activity. In order to minimize the nuclease activity associated with the isolated mt-nucleoids, we isolated the mt-nucleoids from a mutant strain that lacked the mitochondrial nuclease, Nuc1p. In this manner, we succeeded in isolating mt-nucleoids that showed a low level of the nuclease activity. Micrococcal nuclease treatment of these mt-nucleoids led to the continuous digestion of mtDNA in the presence of Ca(2+) ions. MtDNA in the mt-nucleoids also showed the continuous digestion pattern when treated with DNase II. These results suggest that mtDNA in the mt-nucleoids is protected from nuclease digestion by association with proteins, but the organization of the mtDNA-protein complexes is different from that of nuclear chromatin, in which the unit of DNA packaging is regularly repeated.

Use of the nuc1 null mutant for analysis of yeast mitochondrial nucleoids