Kaguni, L. S. | Search bioRxiv

bioRxiv2022

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 [~]55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by [~]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 [~]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 [~]3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.

biophysics↗

bioRxiv2020

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, which in Drosophila melanogaster (Dm) is exceptionally long, 115 amino acids. Molecular modeling predicted this extended linker to fold into a structure similar to the conserved HBD. We measured catalytic activity and substrate binding by EMSA and biolayer interferometry, using a deletion series of protein variants. Both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, whilst loss of the novel HBD led to a [~]90% drop in K[cat] with a decreased K[M], and a large increase in the stability of the RNA/DNA hybrid-enzyme complex. The findings support a bipartite binding model for the enzyme, whereby the second HBD facilitates dissociation of the active site from the product, allowing for processivity. We used shotgun proteomics to identify protein partners of the enzyme involved in mediating these effects. Single-stranded DNA-binding proteins from both the nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, were prominently detected by this method. However, we were not able to document direct interactions between mtSSB and Dm RNase H1 when co-overexpressed in S2 cells, or functional interactions in vitro. Further studies are needed to determine the exact reaction mechanism of Dm RNase H1, the nature of its interaction with mtSSB and the role of the second HBD in both.

molecular biology↗