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Updated: Jun 12, 2026
Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
Published on: June 19, 2018
A single mutation in human mitochondrial DNA polymerase Pol gammaA affects both polymerization and proofreading
Young-Sam Lee1, Kenneth A Johnson, Ian J Molineux
1Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
Abstract:
Common causes of human mitochondrial diseases are mutations affecting DNA polymerase (Pol) gamma, the sole polymerase responsible for DNA synthesis in mitochondria. Although the polymerase and exonuclease active sites are located on the catalytic subunit Pol gammaA, in holoenzyme both activities are regulated by the accessory subunit Pol gammaB. Several patients with severe neurological and muscular disorders were reported to carry the Pol gammaA substitutions R232G or R232H, which lie outside of either active site. We report that Arg(232) substitutions have no effect on independent Pol gammaA activities but show major defects in the Pol gammaA-Pol gammaB holoenzyme, including decreased polymerase and increased exonuclease activities, the latter with decreased selectivity for mismatches. We show that Pol gammaB facilitates distinguishing mismatched from base-paired primer termini and that Pol gammaA Arg(232) is essential for mediating this regulatory function of the accessory subunit. This study provides a molecular basis for the disease symptoms exhibited by patients carrying those substitutions.
Insights
Mutations in mitochondrial DNA polymerase (Pol gamma) cause disease. Substitutions at Arg(232) in Pol gammaA disrupt holoenzyme function, impairing DNA repair and leading to neurological and muscular disorders.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Human mitochondrial diseases are often caused by mutations in DNA polymerase gamma (Pol gamma), the essential enzyme for mitochondrial DNA replication.
- Pol gamma consists of a catalytic subunit (Pol gammaA) and an accessory subunit (Pol gammaB), which regulates its activity.
- Specific Pol gammaA substitutions (R232G/H) found in patients with neurological and muscular disorders are located outside the enzyme's active sites.
Purpose of the Study:
- To investigate the functional impact of Pol gammaA Arg(232) substitutions on the Pol gammaA-Pol gammaB holoenzyme.
- To elucidate the molecular mechanism by which these substitutions contribute to mitochondrial disease pathology.
Main Methods:
- Assessed polymerase and exonuclease activities of wild-type and mutant Pol gammaA and Pol gammaA-Pol gammaB holoenzymes.
- Evaluated the effect of Arg(232) substitutions on the holoenzyme's ability to discriminate between matched and mismatched primer termini.
Main Results:
- Arg(232) substitutions did not affect the independent activities of Pol gammaA.
- In the holoenzyme, these substitutions led to reduced polymerase activity and increased exonuclease activity with diminished mismatch selectivity.
- Pol gammaB's role in distinguishing mismatched from base-paired termini was shown to be dependent on Pol gammaA Arg(232).
Conclusions:
- Pol gammaA Arg(232) is crucial for Pol gammaB's regulatory function in the holoenzyme.
- The identified substitutions disrupt holoenzyme fidelity, providing a molecular explanation for the neurological and muscular symptoms in affected patients.
- This research offers insights into the pathogenesis of mitochondrial DNA polymerase-related disorders.
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