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Updated: Dec 25, 2025
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
Modeling of pathogenic variants of mitochondrial DNA polymerase: insight into the replication defects and implication
Nallely Hoyos-Gonzalez1, Carlos H Trasviña-Arenas1, Andrea Degiorgi2
1Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 629, CP 36821 Irapuato, Guanajuato, Mexico.
Background:
Mutations in human gene encoding the mitochondrial DNA polymerase γ (HsPolγ) are associated with a broad range of mitochondrial diseases. Here we studied the impact on DNA replication by disease variants clustered around residue HsPolγ-K1191, a residue that in several family-A DNA polymerases interacts with the 3' end of the primer.
Methods:
Specifically, we examined the effect of HsPolγ carrying pathogenic variants in residues D1184, I1185, C1188, K1191, D1196, and a stop codon at residue T1199, using as a model the yeast mitochondrial DNA polymerase protein, Mip1p.
Results:
The introduction of pathogenic variants C1188R (yV945R), and of a stop codon at residue T1199 (yT956X) abolished both polymerization and exonucleolysis in vitro. HsPolγ substitutions in residues D1184 (yD941), I1185 (yI942), K1191 (yK948) and D1196 (yD953) shifted the balance between polymerization and exonucleolysis in favor of exonucleolysis. HsPolγ pathogenic variants at residue K1191 (yK948) and D1184 (yD941) were capable of nucleotide incorporation albeit with reduced processivity. Structural analysis of mitochondrial DNAPs showed that residue HsPolγ-N864 is placed in an optimal distance to interact with the 3' end of the primer and the phosphate backbone previous to the 3' end. Amino acid changes in residue HsPolγ-N864 to Ala, Ser or Asp result in enzymes that did not decrease their polymerization activity on short templates but exhibited a substantial decrease for processive DNA synthesis.
Conclusion:
Our data suggest that in mitochondrial DNA polymerases multiple amino acids are involved in the primer-stand stabilization.
Insights
Mutations in mitochondrial DNA polymerase gamma (HsPolγ) cause disease. Variants near K1191 impact DNA replication by altering polymerization and exonucleolysis, highlighting multiple amino acids crucial for primer-strand stabilization.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Mutations in the human mitochondrial DNA polymerase gamma (HsPolγ) gene are linked to various mitochondrial diseases.
- Disease-associated variants often cluster around key functional residues, such as HsPolγ-K1191, which interacts with the primer's 3' end in related DNA polymerases.
Purpose of the Study:
- To investigate the impact of disease-associated variants in HsPolγ on DNA replication fidelity.
- To elucidate the role of specific residues, particularly those near K1191, in the polymerase and exonuclease activities of HsPolγ.
Main Methods:
- Utilized a yeast mitochondrial DNA polymerase (Mip1p) model to study pathogenic variants of HsPolγ, including substitutions at D1184, I1185, C1188, K1191, D1196, and a T1199 stop codon.
- Assessed polymerization and exonuclease activities in vitro.
- Performed structural analysis of mitochondrial DNA polymerases (DNAPs) to understand residue interactions.
Main Results:
- Pathogenic variants C1188R and T1199X abolished both polymerization and exonucleolysis.
- Substitutions at D1184, I1185, K1191, and D1196 shifted the balance towards exonucleolysis.
- Variants at K1191 and D1184 retained nucleotide incorporation but with reduced processivity.
- Changes at residue N864 significantly decreased processive DNA synthesis.
Conclusions:
- Multiple amino acids within mitochondrial DNA polymerases contribute to primer-strand stabilization.
- The findings provide insights into the molecular mechanisms underlying mitochondrial DNA replication defects caused by HsPolγ mutations.
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