Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB.
Nucleic Acids Res
; 51(4): 1750-1765, 2023 02 28.
Article
em En
| MEDLINE
| ID: mdl-36744436
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 â¼55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by â¼30-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 â¼25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by â¼3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Ligação a DNA
/
Replicação do DNA
/
DNA Polimerase gama
Limite:
Humans
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article