title: Purification and Comparative Assay of the Human Mitochondrial Replicative DNA Helicase.
abstract: The replicative mitochondrial DNA (mtDNA) helicase is essential for mtDNA replication and maintenance of the mitochondrial genome. Despite substantial advances that have been made in its characterization, there is still much to be understood about the functional roles of its domains and its interactions with the other components of the minimal mitochondrial DNA replisome. Critical to achieving this is the ability to isolate the enzyme in a stable, active form. In this chapter we describe a modified, streamlined purification strategy for recombinant forms of the enzyme. We also present assays to assess its helix unwinding activity and the stimulatory effects of the mitochondrial single-stranded DNA-binding protein (mtSSB). Finally, we describe a concentration/buffer exchange method that we have employed to achieve greater enzyme stability and appropriate conditions for biochemical and biophysical studies.
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI, 48824, USA.
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Rosado-Ruiz
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI, 48824, USA.
authors.forename: Minyoung
authors.initials: M
authors.lastname: So
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI, 48824, USA. lskaguni@msu.edu.|Institute of Biosciences of Medical Technology, University of Tempere, Tempere, 33014, Finland. lskaguni@msu.edu.
authors.forename: Laurie S
authors.initials: LS
authors.lastname: Kaguni
mesh_terms: D000818:Animals; D002460:Cell Line; D004265:DNA Helicases / Q000235:genetics*; D019915:DNA Primase / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics*; D024101:Mitochondrial Proteins / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D019915:DNA Primase; D004265:DNA Helicases
keywords: Helicases; Human; Mitochondrial DNA replication; Mitochondrial single-stranded DNA-binding protein; mtDNA helicase