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

Document Type

Article

Publication Date

2-28-2023

Subject Area

Humans; DNA Polymerase gamma (metabolism); DNA Replication; DNA, Single-Stranded; DNA-Binding Proteins (metabolism); DNA-Directed DNA Polymerase (metabolism)

Abstract

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, Polγ, and its modulation by cognate and noncognate SSBs. We show that Polγ 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 Polγ 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 Polγ, 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.

Publication Title

Nucleic acids research

Volume

51

Issue

4

First Page

1750

Last Page

1765

Digital Object Identifier (DOI)

10.1093/nar/gkad037

PubMed ID

36744436

E-ISSN

1362-4962

Language

eng

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