Role of Histidine-932 of Human Mitochondrial DNA Polymerase in Nucleotide Discrimination and Inherited Disease

Abstract

The human mitochondrial DNA polymerase (POLG ) is nuclearly-encoded and is solely responsible for the replication and repair of the mitochondrial genome. The progressive accumulation of mutations within the mitochondrial genome is thought to be related to ageing, and mutations in the POLG gene are responsible for numerous heritable disorders including progressive external opthalmoplegia (PEO), Alpers syndrome and Parkinsonism. Here we investigate the kinetic effect of H932Y, a mutation associated with PEO. Mutations H932Y and H932A reduce the specificity constant governing correct nucleotide incorporation 150- and 70-fold respectively, without significantly affecting fidelity of incorporation or the maximum rate of incorporation. However, this leads to only a twofold reduction in rate of incorporation at a physiological nucleotide concentration (∼100 M). Surprisingly, incorporation of T:T or C:T mismatches catalyzed by either H932Y or H932A mutants was followed by slow pyrophosphate release (or fast pyrophosphate rebinding) Also, H932Y readily catalyzed incorporation of multiple mismatches, which may have a profound physiological impact over time. H932 is thought to contact the beta phosphate of the incoming nucleotide, so it is perhaps surprising that H932Y appears to slow rather than accelerate pyrophosphate release.