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Purification and Comparative Assay of the Human Mitochondrial Replicative DNA Helicase

  • Fernando A. Rosado-Ruiz
  • Minyoung So
  • Laurie S. KaguniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1351)

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.

Key words

Mitochondrial DNA replication mtDNA helicase Helicases Mitochondrial single-stranded DNA-binding protein Human 

Notes

Acknowledgements

This work was supported by grant GM45295 from the National Institutes of Health to L.S.K.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Fernando A. Rosado-Ruiz
    • 1
  • Minyoung So
    • 1
  • Laurie S. Kaguni
    • 2
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and MedicineMichigan State UniversityEast LansingUSA
  2. 2.Institute of Biosciences of Medical TechnologyUniversity of TempereTempereFinland

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