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. We present here several methods that allow the identification of the oligomeric state of the human mtDNA helicase, and probe the modular architecture of the enzyme. Despite their relatively common usage, we believe that their versatility makes these techniques particularly helpful in the characterization of oligomeric proteins.