Identification and characterization of a mitochondrial endonuclease from yeast, Schizosaccharomyces pombe.
Schizosaccharomyces pombe mitochondria were isolated from the cells treated with Novozyme 234, and purified in a Percoll gradient. A zymographic assay in a SDS-polyacrylamide gel containing single-stranded DNA revealed that an endonuclease of 32 kDa is associated with the mitochondria. The endonuclease was extracted from the mitochondria with 0.5 M KCl and was partially purified. The 32-kDa enzyme degraded both DNA and RNA at a weak alkaline pH, but preferred single-stranded DNA. The enzyme required Mg2+ or Mn2+, but not Ca2+ or Zn2+ for activity, and was inhibited by 50% with a 150 mM salt solution. Nicks generated by the enzyme could be resealed with T4 DNA ligase, indicating that the enzyme produces 5'-P and 3'-OH ends.
Ikeda
S
S
Department of Biochemistry, Faculty of Science, Okayama University of Science, Japan. ikeda@dbc.ous.ac.jp
Maeda
N
N
Ohshima
T
T
Takata
N
N
D004277:DNA, Single-Stranded / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D012313:RNA / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D012313:RNA; D004720:Endonucleases