Physical and Functional Interaction of Mitochondrial Single-Stranded DNA-Binding Protein and the Catalytic Subunit of DNA Polymerase Gamma

Abstract

The maintenance of the mitochondrial genome depends on a suite of nucleus-encoded proteins, among which the catalytic subunit of the mitochondrial replicative DNA polymerase, Pol γα, plays a pivotal role. Mutations in the Pol γα-encoding gene, POLG, are a major cause of human mitochondrial disorders. Here we present a study of direct and functional interactions of Pol γα with the mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB coordinates the activity of the enzymes at the DNA replication fork. However, the mechanism of this functional relationship is elusive, and no direct interactions between the replicative factors have been identified to date. This contrasts strikingly with the extensive interactomes of SSB proteins identified in other homologous replication systems. Here we show for the first time that mtSSB binds Pol γα directly, in a DNA-independent manner. This interaction is strengthened in the absence of the loop 2.3 structure in mtSSB, and is abolished upon preincubation with Pol γβ. Together, our findings suggest that the interaction between mtSSB and polymerase gamma holoenzyme (Pol γ) involves a balance between attractive and repulsive affinities, which have distinct effects on DNA synthesis and exonucleolysis.

Mitochondrial DNA maintenance in Drosophila melanogaster

“…It also indicates there would always be sufficient Pol γ-β to function in association with Pol γ-α to form the dimeric holoenzyme in a 1:1 configuration for mtDNA replication, in agreement with previously published data [103] and with our speculations on additional roles for the fly Pol γ-β in mitochondrial RNA metabolism. Our data also contrast with data from human tissues, which indicates 2-to 3-fold excess of Pol γ-α over Pol γ-β [43]. PolG1 levels were higher than those of PolG2 (∼62%) only in the ovaries, a tissue that also has elevated relative expression of almost all mtDNA maintenance genes (Figure 2) due to the high levels of mtDNA replication and mitochondrial biogenesis that occur during oogenesis [104,105].…”

“…Another interesting fact about the Pol γ-mtSSB system is that, in humans, in vitro physical interactions between these two proteins appear to occur more prominently in the absence of the Pol γ-β dimer [43]. In addition, human Pol γ-α alone in vitro is an efficient gap-filling polymerase and in fact can participate in mtDNA repair [28].…”

Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB

“…In addition, our results showed that mtSSB decreased the T p ( f ) of the two mitochondrial holoenzymes at a concentration ten-fold lower than that of homologous EcoSSB, whereas noncognate gp2.5 had no significant effects on pause kinetics. These results support again that, even though no physical interactions have been described between Polγ-mtSSB (41), specific functional polymerase-SSB interactions could play a role in the modulation of the equilibrium between the moving and pause states characteristic of each polymerase.…”

“…These results indicate that species-specific interactions between the polymerase and the SSB may also play a role in the coordination of the polymerase and SSB activities at the fork and promotion of pause-free rate of strand displacement DNA synthesis. It is tempting to speculate that these interactions would be likely functional in the case of the mitochondrial polymerase-mtSSB system (41) and physical between the phage holoenzyme and the C-terminal tail of gp2.5 and EcoSSB (74,78). Noteworthy is that the specific interactions also seem to facilitate the inhibition of the pause-free rates of Polγexo- at high mtSSB concentration and tensions, showing the increased sensitivity of this variant to the cognate SSB.…”

“…It binds ssDNA in a sequence independent manner (38,39) and forms the central nucleo-protein complex substrate upon which the mitochondrial polymerase must act (18,40). Although physical interactions between Polγ and mtSSB have not been observed (41), mtSSB can stimulate the primer-extension activity of Polγ (17,42). In other DNA replication systems SSBs have been shown to stimulate the strand displacement activity of DNApols (13,14,33,43,44).…”