Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells.
Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3' blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.
Chattopadhyay
Ranajoy
R
Sealy Center for Molecular Science, Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555-1079, USA.
Wiederhold
Lee
L
Szczesny
Bartosz
B
Boldogh
Istvan
I
Hazra
Tapas K
TK
Izumi
Tadahide
T
Mitra
Sankar
S
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D024101:Mitochondrial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase