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. Substantial amino acid sequence and functional similarities of this novel helicase are shared with the bacteriophage T7 helicase‐primase, a member of the SF‐2 family of helicases. We show in velocity sedimentation and gel filtration analyses that the mitochondrial helicase exists as a hexamer in its native configuration. Limited proteolysis by trypsin results in the production of several stable fragments, and N‐terminal sequencing reveals distinct N‐and C‐terminal polypeptides that represent minimal structural domains. Further analysis of the proteolytic products demonstrates that the termini are not required to maintain a stable oligomeric form, and that the residues responsible reside within amino acids 145‐ ~591. Furthermore, the proteolytic polypeptides that lack their termini retain ATPase activity. The hexameric conformation and modular architecture revealed in our study show that the mitochondrial DNA helicase and bacteriophage T7 helicase‐primase also share structural features. Our findings place the mitochondrial DNA helicase firmly as the newest member of the SF‐2 family of replicative DNA helicases. This research was supported by NIH grant GM45295 to L.S.K.