title: Dynamic effects of cofactors and DNA on the oligomeric state of human mitochondrial DNA helicase.
abstract: 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. 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 nucleoside diphosphate. We find that single-stranded DNA binding occurs in the absence of cofactors and renders the mitochondrial DNA helicase more susceptible to proteolytic digestion. Our studies indicate that the human mitochondrial DNA helicase shares basic properties with the SF4 replicative helicases, but also identify common features with helicases outside the superfamily, including dynamic conformations similar to other AAA(+) ATPases.
authors.affiliation: Department of Biochemistry and Molecular Biology and the Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, Michigan 49924-1319, USA.
authors.forename: Tawn D
authors.initials: TD
authors.lastname: Ziebarth
authors.forename: Rocio
authors.initials: R
authors.lastname: Gonzalez-Soltero
authors.forename: Magdalena M
authors.initials: MM
authors.lastname: Makowska-Grzyska
authors.forename: Rafael
authors.initials: R
authors.lastname: Núñez-Ramírez
authors.forename: Jose-Maria
authors.initials: JM
authors.lastname: Carazo
authors.forename: Laurie S
authors.initials: LS
authors.lastname: Kaguni
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001667:Binding, Competitive; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008928:Mitochondria / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D013816:Thermodynamics
chemical_list: D003432:Cross-Linking Reagents; D004272:DNA, Mitochondrial; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases