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

Abstract

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 Ca²⁺ 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.