Structure-function defects of human mitochondrial DNA polymerase in autosomal dominant progressive external ophthalmoplegia

Abstract

Progressive external ophthalmoplegia (PEO) is a mitochondrial disorder associated with mutations in the POLG gene encoding the mitochondrial DNA polymerase (pol gamma). Four autosomal dominant mutations that cause PEO encode the amino acid substitutions G923D, R943H, Y955C and A957S in the polymerase domain of pol gamma. A homology model of the pol gamma catalytic domain in complex with DNA was developed to investigate the effects of these mutations. Two mutations causing the most severe disease phenotype, Y955C and R943H, change residues that directly interact with the incoming dNTP. Polymerase mutants exhibit 0.03-30% wild-type polymerase activity and a 2- to 35-fold decrease in nucleotide selectivity in vitro. The reduced selectivity and catalytic efficiency of the autosomal dominant PEO mutants predict in vivo dysfunction, and the extent of biochemical defects correlates with the clinical severity of the disease.

Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication

“…Expression and Purification-The WT and mutant forms of the His 6 affinity-tagged recombinant catalytic subunit of human pol ␥ were produced in baculovirus-infected Sf9 cells, and the proteins were purified to homogeneity as described previously (23,24). The His 6 affinity-tagged p55 accessory subunit was expressed in E. coli and purified to homogeneity as described previously (25).…”

Decreased mtDNA, oxidative stress, cardiomyopathy, and death from transgenic cardiac targeted human mutant polymerase γ

“…22 Recombinant Y955C Pol g exhibits o1% wild-type (WT) activity and severely decreased processivity. This stalling of mtDNA synthesis with minimal catalytic activity may explain the profound clinical changes in Y955C heterozygotes, 23 but has not been defined pathophysiologically in a murine transgenic model. Substitution of Y955 to cysteine also increases nucleotide misinsertion errors 1-2 orders of magnitude in absence of exonucleolytic proofreading.…”

“…We published biochemical data on the mutant Y955C Pol g protein in CPEO, 2,22,23,25 however the Y955C POLG mutation has not been investigated in transgenic murine models. The effect of human Y955C Pol g on the murine heart was explored using transgenically targeted Y955C POLG driven by the alpha myosin heavy chain promoter (aMyHC).…”

Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV-infected Zulu population of South Africa

“…Although there is a spectrum in the degree of insufficiency in polymerase activity, the most severe mutations lead to loss of >99% of polymerase function and cell death due to insufficient cellular energy production. [28,29] The pathogenicity of the p.E1143G change, caused by an A-to-G substitution at nucleotide position 3428 in exon 21, has been widely scrutinised. [28,30,31] The substitution is found at a high frequency in control populations (2 -3%), and has never been identified alone in any disease patients.…”

“…[28,29] The pathogenicity of the p.E1143G change, caused by an A-to-G substitution at nucleotide position 3428 in exon 21, has been widely scrutinised. [28,30,31] The substitution is found at a high frequency in control populations (2 -3%), and has never been identified alone in any disease patients. [32] E1143 is a highly conserved amino acid located adjacent to motif C of the polymerase domain, and biochemical investigations show a possible role in the modular expression of other POLG1 mutations in cis.…”