Identification and characterization of a mitochondrial endonuclease from yeast, Schizosaccharomyces pombe

Biochem Mol Biol Int. 1996 Nov;40(5):1017-24. doi: 10.1080/15216549600201653.

Abstract

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.

MeSH terms

  • DNA, Single-Stranded / metabolism
  • Endonucleases / chemistry
  • Endonucleases / isolation & purification*
  • Endonucleases / metabolism
  • Hydrogen-Ion Concentration
  • Mitochondria / enzymology*
  • Molecular Weight
  • RNA / metabolism
  • Schizosaccharomyces / enzymology*
  • Substrate Specificity

Substances

  • DNA, Single-Stranded
  • RNA
  • Endonucleases