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

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

Author information

1
From the Institute of Biosciences and Medical Technology, University of Tampere, 33520 Tampere, Finland, the Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, Michigan 48823, and.
2
the Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27514.
3
the Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, Michigan 48823, and.
4
From the Institute of Biosciences and Medical Technology, University of Tampere, 33520 Tampere, Finland, the Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, Michigan 48823, and lskaguni@msu.edu.

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)

PMID:
26446790
PMCID:
PMC4661385
DOI:
10.1074/jbc.M115.673707
[Indexed for MEDLINE]
Free PMC Article
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