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

Many replicative DNA polymerases couple DNA replication and unwinding activities to perform strand displacement DNA synthesis, a critical ability for DNA metabolism. Strand displacement is tightly regulated by partner proteins, such as single-stranded DNA (ssDNA) binding proteins (SSBs) by a poorly understood mechanism. Here, we use single-molecule optical tweezers and biochemical assays to elucidate the molecular mechanism of strand displacement DNA synthesis by the human mitochondrial DNA polymerase, Pol gamma, and its modulation by cognate and noncognate SSBs. We show that Pol gamma exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by similar to 55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by similar to 30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Pol gamma strand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by similar to 25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Pol gamma, in turn promoting the productive polymerization state by similar to 3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.

Automated summary

In this study, the molecular mechanism of strand displacement DNA synthesis by human mitochondrial DNA polymerase Pol gamma and its modulation by single-stranded DNA binding proteins (SSBs) were elucidated using single-molecule optical tweezers and biochemical assays. The results showed that SSBs stimulate Pol gamma's strand displacement activity through multiple mechanisms, playing a significant role in the replication of human mitochondrial DNA.