Synergistic Effects of thein cisT251I and P587L Mitochondrial DNA Polymerase γ Disease Mutations.
Human mitochondrial DNA (mtDNA) polymerase γ (Pol γ) is the only polymerase known to replicate the mitochondrial genome. The Pol γ holoenzyme consists of the p140 catalytic subunit (POLG) and the p55 homodimeric accessory subunit (POLG2), which enhances binding of Pol γ to DNA and promotes processivity of the holoenzyme. Mutations withinPOLGimpede maintenance of mtDNA and cause mitochondrial diseases. Two commonPOLGmutations usually foundin cisin patients primarily with progressive external ophthalmoplegia generate T251I and P587L amino acid substitutions. To determine whether T251I or P587L is the primary pathogenic allele or whether both substitutions are required to cause disease, we overproduced and purified WT, T251I, P587L, and T251I + P587L double variant forms of recombinant Pol γ. Biochemical characterization of these variants revealed impaired DNA binding affinity, reduced thermostability, diminished exonuclease activity, defective catalytic activity, and compromised DNA processivity, even in the presence of the p55 accessory subunit. However, physical association with p55 was unperturbed, suggesting intersubunit affinities similar to WT. Notably, although the single mutants were similarly impaired, a dramatic synergistic effect was found for the double mutant across all parameters. In conclusion, our analyses suggest that individually both T251I and P587L substitutions functionally impair Pol γ, with greater pathogenicity predicted for the single P587L variant. Combining T251I and P587L induces extreme thermal lability and leads to synergistic nucleotide and DNA binding defects, which severely impair catalytic activity and correlate with presentation of disease in patients.
From the Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Karen L
KL
DeBalsi
From the Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Matthew J
MJ
Longley
From the Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Kirsten E
KE
Hoff
From the Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 copelan1@niehs.nih.gov.
William C
WC
Copeland
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D000074002:DNA Polymerase gamma; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology*; D028361:Mitochondrial Diseases / Q000235:genetics / Q000473:pathology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D004272:DNA, Mitochondrial; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human
DNA polymerase; DNA replication; mitochondrial DNA (mtDNA); mitochondrial DNA damage; mitochondrial DNA mutations; mitochondrial DNA replication; mitochondrial disease; progressive external ophthalmoplegia