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Purification and Comparative Assay of Human Mitochondrial Single-Stranded DNA-Binding Protein

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

Abstract

The mitochondrial single-stranded DNA-binding protein (mtSSB) coordinates the function of replisome components at the mitochondrial replication fork. In recent years, it has been demonstrated that mtSSB stimulates the activities of DNA polymerase γ (Pol γ) and mitochondrial DNA (mtDNA) helicase in a concentration-dependent manner. Here we present a new approach to purify the human mtSSB and our standard assays to evaluate its biochemical properties, including a Gel Mobility Shift Assay (GMSA) to assess single-stranded DNA (ssDNA) binding activity, and an assay to assess SSB stimulation of Pol γ activity.

Key words

Mitochondrial DNA replication Mitochondrial single-stranded DNA-binding protein DNA polymerase γ Human 

Notes

Acknowledgments

This work was supported by grant GM45295 from the National Institutes of Health and funds from the University of Tampere to L.S.K. G.C. was supported in part by Biocenter Finland.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

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

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