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.