Mitochondrial factors modulate the activity of endonuclease G, the major nuclease of Mammalian mitochondria.
Endonuclease G (Endo G), a sugar-nonspecific nuclease preferring single-stranded DNA (ssDNA), is responsible for major nuclease activity in mitochondria. If the enzyme provides an important nicking function for mitochondrial DNA (mtDNA) in vivo, then mitochondrial factors likely exist which modulate the enzyme's activity and prevent cleavage at single-stranded moieties of mtDNA. In the present paper, we report that specific membrane phospholipids, polyamines and single-stranded DNA-binding protein (SSB) appear to exert such effects in vitro. Phosphatidylcholine and phosphatidylethanolamine, the major constituents of the mitochondrial inner membrane, stimulated purified Endo G activity 5- to 10-fold. Spermine at 5-100 microM also stimulated activity about 4-fold. However, at more than 500 microM, the spermine largely inhibited the degradation of ssDNA and duplex DNA. Escherichia coli SSB, which has physicochemical properties analogous to those of mitochondrial SSB (mtSSB), markedly inhibited the degradation of phiX174 ssDNA by Endo G, indicating the possible involvement of mtSSB in vivo in protection of single-stranded regions of mtDNA from nucleolytic attacks.
Ikeda
Shogo
S
Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridaicho, Japan. ikeda@dbc.ous.ac.jp
Seki
Yuichi
Y
Ozaki
Kohji
K
D000818:Animals; D010584:Bacteriophage phi X 174 / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D066298:In Vitro Techniques; D008081:Liposomes / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology*; D009206:Myocardium / Q000166:cytology / Q000201:enzymology; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D013096:Spermine / Q000378:metabolism
D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D013096:Spermine; D004706:Endodeoxyribonucleases; C052391:endonuclease G