Mitochondrial endonuclease activities specific for apurinic/apyrimidinic sites in DNA from mouse cells.
Endonuclease activity which specifically cleaves baseless (apurinic/apyrimidinic (AP] sites in supercoiled DNA has been purified from mitochondria of the mouse plasmacytoma cell line, MPC-11. Two variant forms separate upon purification; these have small but reproducible differences in catalytic and chromatographic properties, but similar physical properties. Both have a sedimentation coefficient of 4.0, corresponding to a molecular weight of 61,000 (assuming a globular configuration) and a peptide molecular weight of about 65,000 as determined by immunoblot analysis with antiserum raised against the major AP endonuclease from HeLa cells. Thus mitochondrial AP endonuclease appears to be a monomer of about 65 kDa, making it distinguishable from the major AP endonuclease of MPC-11 cells which, like those of other mammalian cells, appears to be a monomer of about 41 kDa. A possible 82-kDa precursor form was also detected by immunoblot analysis of a crude mitochondrial fraction. Mitochondrial AP endonuclease activity is greatly stimulated by divalent cations, has a pH optimum between 6.5 and 8.5, and cleaves the AP site by a class II mechanism to generate a 3'-OH nucleotide residue. These properties resemble those of the major mammalian AP endonucleases but, unlike those enzymes, mitochondrial AP endonuclease activity is neither inhibited by adenine or NAD+ nor stimulated by Triton X-100. Since the mitochondrial activity generates active primer termini for DNA synthesis, it could function in base excision DNA repair; alternatively, it might have a role in eliminating damaged mitochondrial genomes from the gene pool.
Tomkinson
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AE
Department of Biochemistry, University of California, Berkeley 94720.
Bonk
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RT
Linn
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S
D001080:Apurinic Acid / Q000378:metabolism*; D002413:Cations, Divalent; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D010954:Plasmacytoma; D011119:Polynucleotides / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D002413:Cations, Divalent; D004278:DNA, Superhelical; D011119:Polynucleotides; C001790:apyrimidinic acid; D001080:Apurinic Acid; D004247:DNA; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase