Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos.
A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE-cellulose and heparin--avidgel-F, followed by FPLC chromatography on mono S, Superose 12 and a second mono S column. This enzyme digests double-stranded DNA more efficiently than heat-denatured DNA. The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel-filtration Superose 12 column. The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein. The enzyme has an absolute requirement for divalent cations, preferring Mg2+ over Mn2+. No activity could be detected when these cations were replaced by Ca2+ or Zn2+. The pH optimum for this enzyme activity is 6.5-7.4 and its isoelectric point is 4.9. Both single-strand and double-strand breaks are introduced simultaneously into a supercoiled substrate in the presence of MgCl2 or MnCl2. Endonuclease-treated DNA serves as a substrate for DNA polymerase I from Escherichia coli, suggesting that 3'-OH termini are generated during cleavage. The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity. Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharomyces cerevisiae have revealed a potential structural homology between these nucleases.
Harosh
I
I
Department of Genetics, University of California, Davis.
Mezzina
M
M
Harris
P V
PV
Boyd
J B
JB
D000818:Animals; D002413:Cations, Divalent; D002845:Chromatography; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004331:Drosophila melanogaster / Q000196:embryology* / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity
D002413:Cations, Divalent; D004278:DNA, Superhelical; D008345:Manganese; D004247:DNA; D004720:Endonucleases; D008274:Magnesium