Stimulation of Variant Forms of the Mitochondrial DNA Helicase Twinkle by the Mitochondrial Single-Stranded DNA-Binding Protein

Methods Mol Biol. 2021:2281:313-322. doi: 10.1007/978-1-0716-1290-3_20.

Abstract

Defects in mitochondrial DNA (mtDNA) maintenance may lead to disturbances in mitochondrial homeostasis and energy production in eukaryotic cells, causing diseases. During mtDNA replication, the mitochondrial single-stranded DNA-binding protein (mtSSB) stabilizes and protects the exposed single-stranded mtDNA from nucleolysis; perhaps more importantly, it appears to coordinate the actions of both the replicative mtDNA helicase Twinkle and DNA polymerase gamma at the replication fork. Here, we describe a helicase stimulation protocol to test in vitro the functional interaction between mtSSB and variant forms of Twinkle. We show for the first time that the C-terminal tail of Twinkle is important for such an interaction, and that it negatively regulates helicase unwinding activity in a salt-dependent manner.

Keywords: Mitochondrial DNA replication; P66; Twinkle; dsDNA unwinding assay; mtSSB.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • DNA Helicases / chemistry*
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA Replication
  • DNA, Mitochondrial / chemistry
  • DNA, Mitochondrial / metabolism
  • DNA, Single-Stranded / chemistry
  • DNA, Single-Stranded / metabolism*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • Humans
  • Mitochondrial Proteins / chemistry*
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*
  • Models, Molecular
  • Mutation*
  • Protein Binding
  • Protein Conformation

Substances

  • DNA, Mitochondrial
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Mitochondrial Proteins
  • SSBP1 protein, human
  • DNA Helicases
  • TWNK protein, human