RT Journal Article
T1 Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB
A1 Plaza-G.A., Ismael
A1 Lemishko, Kateryna M.
A1 Crespo, Rodrigo
A1 Truong, Thinh Q.
A1 Kaguni, Laurie S.
A1 Cao García, Francisco Javier
A1 Ciesielski, Grzegorz L.
A1 Ibarra, Borja
AB 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.
PB Oxford University Press (OUP)
SN 0305-1048
YR 2023
FD 2023-02-06
LK https://hdl.handle.net/20.500.14352/104344
UL https://hdl.handle.net/20.500.14352/104344
LA eng
NO Ismael Plaza-G.A., Kateryna M Lemishko, Rodrigo Crespo, Thinh Q Truong, Laurie S Kaguni, Francisco J Cao-García, Grzegorz L Ciesielski, Borja Ibarra, Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB, Nucleic Acids Research, Volume 51, Issue 4, 28 February 2023, Pages 1750–1765, https://doi.org/10.1093/nar/gkad037
NO Ministerio de Economía y Competitividad (España)
NO National Institutes of Health
NO Comunidad de Madrid
NO Ministerio de Ciencia, Innovación y Universidades (España)
NO University of Alabama
NO Universitat Autònoma de Barcelona
DS Docta Complutense
RD 24 abr 2025