Dynamic Effects of Cofactors and DNA on the Oligomeric State of the Human Mitochondrial DNA Helicase

Abstract

Mitochondria are the essential energy‐producing organelle in eukaryotes, and represent the only organelle in animals that contains its own genome. Maintenance of the mitochondrial DNA (mtDNA) genome is dependent on myriad nuclear‐encoded proteins including the mtDNA helicase, which is an essential component of the replicative machinery. Human mtDNA helicase shares a high degree of sequence similarity with the bacteriophage T7 primase‐helicase gene 4 protein, and catalyzes duplex unwinding in the 5’‐3’ direction. We have shown that it is an oligomer in solution that is destabilized under low salt conditions. We present here the effects of cofactors on its stability and oligomerization state. We show that the low salt sensitivity of the mtDNA helicase is alleviated by the presence of magnesium, nucleotide and increased temperature. Electron microscopic and glutaraldehyde cross‐linking analyses provide the first evidence of a heptameric oligomer, and its interconversion from a hexameric form. Limited proteolysis by trypsin shows that binding of nucleoside triphosphate produces a conformational change that is distinct from the conformation observed in the presence of nucleotide diphosphate. We find that single‐stranded DNA binding occurs in the absence of cofactors, and renders the mtDNA helicase more susceptible to proteolytic digestion. Experiments to examine oligomeric state upon DNA binding are underway to evaluate the functional form of the enzyme, and its dynamic changes along the reaction pathway. This research was supported by NIH grant GM45295.