Human mitochondrial DNA polymerase holoenzyme: reconstitution and characterization

Area of Science:

• Molecular Biology
• Biochemistry
• Enzymology

Background:

• Human mitochondrial DNA polymerase is crucial for maintaining mitochondrial genome stability.
• The holoenzyme's function, particularly the role of its accessory subunit, requires detailed investigation.
• Understanding this enzyme is vital for developing antiviral nucleoside analogues.

Purpose of the Study:

• To reconstitute and characterize the human mitochondrial DNA polymerase holoenzyme.
• To elucidate the specific functions of the accessory subunit in DNA polymerization.
• To establish kinetic parameters for the catalytic subunit alone versus the holoenzyme.

Main Methods:

• Cloning and overexpression of catalytic and accessory subunits.
• Reconstitution of the two-subunit human mitochondrial DNA polymerase holoenzyme.
• Kinetic analysis of DNA binding, nucleotide binding, and DNA polymerization rates.

Main Results:

• The accessory subunit binds the catalytic subunit with a dissociation constant of 35 +/- 16 nM.
• The accessory subunit significantly enhances DNA binding (3.5-fold tighter), nucleotide binding (improved from 4.7 to 0.78 μM), and polymerization rate (increased from 8.7 to 45 s⁻¹).
• Processivity increases dramatically from ~290 to ~2250 units with the accessory subunit.

Conclusions:

• The accessory subunit is essential for optimal human mitochondrial DNA polymerase function, profoundly impacting multiple kinetic parameters.
• The term "processivity factor" inadequately describes the accessory subunit's broad stimulatory effects on nucleotide binding and incorporation.
• The reconstituted holoenzyme provides a platform for studying enzyme specificity and the mechanisms of antiviral nucleoside analogue toxicity.