The role of residue Y955 of mitochondrial DNA polymerase [gamma] in nucleotide binding and discrimination

Abstract

The human mitochondrial polymerase (pol γ) is a nuclearly-encoded polymerase that is solely responsible for the faithful replication and repair of the mitochondrial genome. The Y955C mutation in pol γ results in early onset progressive external ophthalmoplegia, premature ovarian failure, and Parkinson’s disease. It is believed that the position of this Y955 residue on the catalytic helix in the polymerase makes it responsible for stabilizing the incoming nucleotide. I have investigated the kinetic effect of the Y955C mutation. Mutation of the tyrosine to a cysteine resulted in a decreased maximum rate of polymerization and increased the dissociation constant for incoming nucleotide. In turn, this decreased catalytic efficiency by 30 to 100-fold. In addition, the polymerase did not incorporate all bases with the same efficiency, it was most efficient when incorporating dGTP opposite a dC, but showed less efficient catalysis when faced with an A:T or T:A base-pair. The polymerase also showed reduced discrimination against misincorporation events. However, when presented with an oxidatively-damaged base, 8-oxo-deoxyguanosine, the polymerase chose to incorporate the base in the correct conformation opposite a dC, discriminating against the mutagenic incorporation of 8-oxo-dGTP opposite a dA. The results presented in this thesis suggest that the severe clinical symptoms of patients with this mutation are at least due in part to the reduced efficiency and discrimination of this polymerase γ mutation.