Despite the ubiquitous trend of increasing complexity in eukaryotes, the vital process of mtDNA replication has retained a simplistic, three component replisome consisting of the mtDNA Pol γ, mtSSB and the mtDNA replicative helicase. This minimal mitochondrial replisome resembles the bacteriophage T7 replisome and may even be simpler due to the apparent absence of a primase function in the N-terminal domain of the mtDNA replicative helicase. Interestingly, insects have retained the four cysteines of motif I which have been shown to bind zinc in T7, so an N-terminal construct (NTD) of Drosophila melanogaster mtDNA replicative helicase was designed containing the conserved residues. Purified fractions of the NTD construct contain an iron sulfur cluster, as determined by UV-Visible spectroscopy, as well as iron and sulfide determination. This work describes for the first time an iron sulfur cluster site in dm-mtDNA replicative helicase and evidence of weak DNA binding by the NTD using a FRET assay. Mutations in Pol γ represent a major cause of human mitochondrial diseases, especially those affecting the nervous system in adults and in children. More than 160 POLG1 disease mutations have been identified, which are nearly uniformly distributed along the length of the POLG1 sequence. Comprehensive literature analysis of the biochemical properties and disease characteristics of Pol γ in light of the crystal structure have revealed genotype-phenotype correlations that support the clustering of mutations into five functional modules in the catalytic core of Pol γ. Our results suggest that cluster prediction can be used to evaluate both the likely biochemical defects and the relative pathogenicity of new POLG variants.
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