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Identification and characterization of a mitochondrial endonuclease from yeast, Schizosaccharomyces pombe
Author(s) -
Ikeda Shogo,
Maeda Noriaki,
Ohshima Tomoaki,
Takata Norifumi
Publication year - 1996
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216549600201653
Subject(s) - schizosaccharomyces pombe , endonuclease , biochemistry , enzyme , dna ligase , microbiology and biotechnology , dna , yeast , biology , schizosaccharomyces , gel electrophoresis , polyacrylamide gel electrophoresis , percoll , chemistry , saccharomyces cerevisiae , in vitro
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 KCI 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.