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

Abstract

Background: mtSSB stimulates the activity of Pol ␥. Results: Stimulation of Pol ␥ activity by SSB correlates with the organization of ssDNA templates in a species-independent manner. Conclusion: Organization of the template DNA by mtSSB is the major factor contributing to the stimulation of Pol ␥ activity. Significance: This study provides insight into the functional relationship of Pol ␥ and mtSSB and a general mechanism for it.

Animal Mitochondrial DNA Replication

“…However, the stimulation profile is mtSSB specific: both Pol γs reach their maximal activity at a concentration of the insect mtSSB lower than that required in the presence of human mtSSB. This corresponds to a similar shift in the concentrations required for generation of specific mtSSB-template complexes (Figure 6D) that correlate with individual phases of the stimulatory effect [48]. That the ssDNA binding affinity of both mtSSBs are similar suggests the more efficient DNA template organization by D. melanogaster mtSSB compensates for the lower DNA synthetic capacity of D. melanogaster Pol γ.…”

“…At the mtDNA replication fork, mtSSB most likely coordinates interactions within the replisome, stimulating Pol γ and mtDNA helicase function. In vitro stimulation of the DNA polymerase and exonuclease activities of Pol γ by mtSSB has been documented in both the human and D. melanogaster systems [44–48]. Human mtSSB stimulates both the unwinding activity of the human mtDNA helicase up to 8-fold [46, 47, 49], and the concerted actions of the human Pol γ and mtDNA helicase in strand-displacement DNA synthesis [50].…”

“…We reported recently a direct comparison of recombinant Pol γ holoenzymes from a vertebrate (human) and an insect ( D. melanogaster ) species, which showed that the extent of DNA synthesis on a singly-primed, circular ssDNA template is ~5-fold higher with the human enzyme [48] (Figure 6C). In comparison, when variants of the human and mouse Pol γ-β that are unable to homodimerize were examined in similar in vitro assays in a heterodimeric state with Pol γ-α, thus resembling insect Pol γ, the decrease in DNA polymerase activity observed as compared to that of their native heterotrimeric forms ranged from ~4- to 7-fold [65, 68, 69].…”

“…Indeed, mtSSB has been shown to stimulate both the DNA polymerase and exonuclease activities of Pol γ ~20-fold, although this value varies substantially between the human and D. melanogaster systems [44–47]. To explore this, we pursued a comparative analysis of the human and D. melanogaster Pol γ holoenzymes with their cognate and heterologous mtSSBs using a combination of biochemical assays and transmission electron microscopy [48]. Under optimal conditions for each, both holoenzymes showed comparable DNA synthesis in the presence of their cognate SSBs.…”

“…In fact, loop 2,3 and α-helix 1 show little sequence similarity between vertebrate and invertebrate species, and an extension of 6–7 amino acid residues in loop 2,3, which is disordered in the crystal structure of the human mtSSB, appears predominantly in vertebrate species (Figure 4A). Our recent electron microscopic study shows that loop 2,3 may be important for human mtSSB to organize the template in a competent configuration for Pol γ [48]. The extended loop 2,3 of vertebrate mtSSB, together with the presence of the additional Pol γ-β protomer in the vertebrate mitochondrial replicase, and other putative structural evolutionary novelties described for the catalytic subunit [18] may then explain the biochemical differences in the vertebrate and insect systems.…”