Functional Roles of the N- and C-Terminal Regions of the Human Mitochondrial Single-Stranded DNA-Binding Protein

Abstract

Biochemical studies of the mitochondrial DNA (mtDNA) replisome demonstrate that the mtDNA polymerase and the mtDNA helicase are stimulated by the mitochondrial single-stranded DNA-binding protein (mtSSB). Unlike Escherichia coli SSB, bacteriophage T7 gp2.5 and bacteriophage T4 gp32, mtSSBs lack a long, negatively charged C-terminal tail. Furthermore, additional residues at the N-terminus (notwithstanding the mitochondrial presequence) are present in the sequence of species across the animal kingdom. We sought to analyze the functional importance of the N- and C-terminal regions of the human mtSSB in the context of mtDNA replication. We produced the mature wild-type human mtSSB and three terminal deletion variants, and examined their physical and biochemical properties. We demonstrate that the recombinant proteins adopt a tetrameric form, and bind single-stranded DNA with similar affinities. They also stimulate similarly the DNA unwinding activity of the human mtDNA helicase (up to 8-fold). Notably, we find that unlike the high level of stimulation that we observed previously in the Drosophila system, stimulation of DNA synthesis catalyzed by human mtDNA polymerase is only moderate, and occurs over a narrow range of salt concentrations. Interestingly, each of the deletion variants of human mtSSB stimulates DNA synthesis at a higher level than the wild-type protein, indicating that the termini modulate negatively functional interactions with the mitochondrial replicase. We discuss our findings in the context of species-specific components of the mtDNA replisome, and in comparison with various prokaryotic DNA replication machineries.

Human Mitochondrial DNA Helicase TWINKLE Is Both an Unwinding and Annealing Helicase

“…5A). It has been shown that mtSSB stimulates unwinding by TWINKLE by almost 8-fold in a helicase assay similar to the M13-based unwinding assay that we have used here (24). Because the heterologous T7 gp2.5 stimulates the helicase activity of TWINKLE, it appears that the stimulation by single strand-binding proteins is not protein-specific.…”

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

“…A 15-mer oligodeoxynucleotide complementary to M13 DNA was synthesized in an Applied Biosystems oligonucleotide synthesizer and then used to prepare the singly primed M13 DNA for DNA polymerase assays as described previously (18). The 48-mer oligodeoxynucleotide used in gel mobility shift assays was as described previously (19).…”

Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

“…Among these proteins, only SSB has been clearly identified in mitochondria. The mitochondrial SSB has a prokaryotic origin and exists in a homotetramer form in both yeast and human (93)(94)(95)(96). Yeast mitochondria also harbor the well-defined Holliday junction resolvase Mgt1/Cce1 (see above).…”

“…This phenotype is suppressed by overexpressing the mitochondrial single-strand binding protein Rim1 (93). Mitochondrial SSB is known to stimulate DNA helicase activity (96). Indeed, Rim1 stimulates Pif1 activity by 4-to 5-fold, and these two proteins physically interact with each other (103).…”