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

Grzegorz L Ciesielski,Laurie S Kaguni,Carolina De Bovi Pontes,Shalom Kim

doi:10.3389/fgene.2021.721864

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.

Highlights

Replication of the mitochondrial genome depends on a set of nucleus-encoded proteins (Ciesielski et al, 2016b)
The synthesis of mitochondrialDNA is catalyzed by the DNA polymerase gamma holoenzyme (Pol γ), which in vertebrates comprises the catalytic subunit, Pol γα, and a dimeric accessory subunit, Pol γβ2 (Kaguni, 2004; Ciesielski et al, 2016b)
The specificity of the binding can be inferred from the fact that it occurs in spite of the presence of excess bovine serum albumin (BSA), which alone exhibited no affinity for the functionalized sensors (Figure 2A)

Summary

Introduction

Replication of the mitochondrial genome depends on a set of nucleus-encoded proteins (Ciesielski et al, 2016b). Whereas bacterial homologues have been demonstrated to play a direct role in the recruitment of the genome replication factors, no such physical interactions of mtSSB have been documented (Oliveira and Ciesielski, 2021). In the absence of the putative interaction, the DNA synthesis rate of Pol γ is significantly reduced (Cerrón et al, 2019) These results suggest that mtSSB may interact with replication factors directly, despite the lack of the C-terminus

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Conclusion