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Mitochondrial endonuclease activities specific for apurinic/apyrimidinic sites in DNA from mouse cells
A E Tomkinson1, R T Bonk, S Linn
1Department of Biochemistry, University of California, Berkeley 94720.
Abstract:
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.
Insights
Mitochondrial AP endonuclease, an enzyme that repairs DNA, was purified from mouse cells. This enzyme differs from its nuclear counterpart and may play a role in DNA repair or mitochondrial genome maintenance.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Mitochondria possess unique DNA repair mechanisms.
- Apurinic/apyrimidinic (AP) sites are common DNA lesions requiring specific endonuclease activity for repair.
- Understanding mitochondrial DNA repair is crucial for cellular health and disease research.
Purpose of the Study:
- To purify and characterize the AP endonuclease activity from mouse plasmacytoma MPC-11 cell mitochondria.
- To compare the properties of mitochondrial AP endonuclease with its nuclear counterpart.
- To elucidate the potential role of mitochondrial AP endonuclease in DNA repair and genome maintenance.
Main Methods:
- Purification of endonuclease activity from MPC-11 cell mitochondria.
- Biochemical characterization including sedimentation analysis, molecular weight determination (SDS-PAGE, immunoblotting), and enzymatic assays.
- Analysis of catalytic properties such as pH optimum, divalent cation dependence, and substrate cleavage mechanism.
Main Results:
- Two distinct forms of mitochondrial AP endonuclease were purified with similar physical but different catalytic and chromatographic properties.
- The enzyme appears as a monomer of approximately 65 kDa, distinct from the 41 kDa nuclear AP endonuclease.
- Mitochondrial AP endonuclease activity is stimulated by divalent cations, has a pH optimum of 6.5-8.5, and functions via a class II mechanism, generating a 3'-OH terminus.
- Unlike nuclear AP endonucleases, the mitochondrial enzyme is not inhibited by adenine or NAD+ and not stimulated by Triton X-100.
- A potential 82-kDa precursor form was detected in crude mitochondrial fractions.
Conclusions:
- Mouse mitochondrial AP endonuclease is a distinct enzyme from its nuclear counterpart, characterized by its size and unique regulatory properties.
- The enzyme's ability to generate primer termini suggests a role in base excision DNA repair within mitochondria.
- Mitochondrial AP endonuclease may also contribute to the elimination of damaged mitochondrial DNA, preserving the integrity of the mitochondrial genome.