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

The Journal of General and Applied Microbiology

Yu Kadowaki¹  Isamu Miyakawa¹  Ryoko Fujimura¹ 

[1] Department of Physics, Biology, and Informatics, Faculty of Science, Yamaguchi University

关键词: micrococcal nuclease;    mitochondrial nucleoids;    NUC1 nuclease;    Saccharomyces cerevisiae;    yeast;   

DOI  :  10.2323/jgam.54.317

学科分类：微生物学和免疫学

来源: Applied Microbiology, Molecular and Cellulrar Biosciences Research Foundation

【 摘 要 】

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.

【 授权许可】

Unknown