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DNA topoisomerase I from calf thymus mitochondria is associated with a DNA binding, inner membrane protein

J H Lin1, G M Lazarus, F J Castora

  • 1Department of Biochemistry, Eastern Virginia Medical School, Norfolk 23501.

Archives of Biochemistry and Biophysics
|March 1, 1992
PubMed

Insights

Mitochondrial DNA topoisomerase activity involves two polypeptides, p78 and p63. The catalytic activity is solely in p78, which functions alone or as a heterodimer with p63.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Mitochondrial DNA topoisomerase is crucial for DNA replication and repair.
  • Two polypeptides, p78 and p63, co-purify with this enzymatic activity.
  • Their specific roles and relationship were previously undefined.

Purpose of the Study:

  • To characterize the relationship between p78 and p63 in mitochondrial DNA topoisomerase.
  • To elucidate the functional roles of each polypeptide in enzymatic activity.
  • To determine if the polypeptides are structurally related.

Main Methods:

  • Enzyme purification from calf thymus mitochondria.
  • Assays for DNA topoisomerase activity (supercoil relaxation, covalent DNA-enzyme linkage).
  • Proteolytic digestion (V8 protease) and fragment analysis.
  • Analysis of enzyme activity under varying ionic strengths and salt concentrations.

Main Results:

  • Mitochondrial topoisomerase functions as both a free monomer and a heterodimer.
  • Catalytic activity resides exclusively in the p78 polypeptide.
  • p78 alone is sufficient for activity at low ionic strength.
  • A 140-kDa heterodimer of p78 and p63 exists in high salt conditions.
  • V8 protease digestion yielded distinct fragment patterns for p78 and p63, indicating no structural relation.

Conclusions:

  • The p78 polypeptide is the catalytic subunit of mitochondrial DNA topoisomerase.
  • The p63 polypeptide is not catalytically active but forms a heterodimer with p78.
  • p63 likely plays a regulatory or structural role in mitochondrial topoisomerase function.
  • The distinct proteolytic profiles suggest p63 is not a precursor or degradation product of p78.

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