Physical analysis of recombinant forms of the human mitochondrial DNA helicase.

Maintenance of the mitochondrial DNA (mtDNA) genome is dependent on numerous 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. As purified at 300 mM NaCl, the enzyme exists as a hexamer, with a modular architecture comprising distinct N- and C-terminal domains.

Dynamic effects of cofactors and DNA on the oligomeric state of human mitochondrial DNA helicase.

We examined the effects of cofactors and DNA on the stability, oligomeric state and conformation of the human mitochondrial DNA helicase. We demonstrate that low salt conditions result in protein aggregation that may cause dissociation of oligomeric structure. The low salt sensitivity of the mitochondrial DNA helicase is mitigated by the presence of magnesium, nucleotide, and increased temperature.

Purification strategy for recombinant forms of the human mitochondrial DNA helicase.

In this chapter, we present a streamlined purification for the production of near-homogeneous and high yield recombinant forms of the human mitochondrial DNA helicase. Minimizing the number of steps and the time elapsed for purification of this enzyme facilitates studies of its structure and mechanism and allows elucidation of native features of both wild-type- and human disease-related forms.

Modular architecture of the hexameric human mitochondrial DNA helicase.

We have probed the structure of the human mitochondrial DNA helicase, an enzyme that uses the energy of nucleotide hydrolysis to unwind duplex DNA during mitochondrial DNA replication. This novel helicase shares substantial amino acid sequence and functional similarities with the bacteriophage T7 primase-helicase. We show in velocity sedimentation and gel filtration analyses that the mitochondrial DNA helicase exists as a hexamer.