Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

J Biol Chem. 2015 Nov 27;290(48):28697-707. doi: 10.1074/jbc.M115.673707. Epub 2015 Oct 7.

Abstract

The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems.

Keywords: DNA polymerase; DNA replication; electron microscopy (EM); enzyme mechanism; mitochondrial DNA (mtDNA).

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • DNA Polymerase gamma
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster
  • Humans
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*

Substances

  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Drosophila Proteins
  • Mitochondrial Proteins
  • SSBP1 protein, human
  • mtSSB protein, Drosophila
  • DNA Polymerase gamma
  • DNA-Directed DNA Polymerase