DNA replication machinery: Insights from in vitro single-molecule approaches

Bocanegra, Rebeca; Płaza, G. A.; Pulido, C. R.; Ibarra, Borja

doi:10.1016/j.csbj.2021.04.013

“…We used a miniaturized counter propagating dual-beam optical tweezers instrument ( 44 ) to manipulate individual DNA hairpins tethered between a streptavidin-coated bead (2.1 μm, Kisker Biotech) immobilized on top of a micropipette and an anti-digoxigenin-coated bead (3.0 μm diameter, Kisker Biotech) held in the optical trap, Figure 1B ( 45 ). Proteins were introduced inside the flow cell after dilution in the replication buffer containing 50 mM Tris pH 8.5, 30 mM KCl, 10 mM DTT, 4 mM MgCl 2 , 0.2 mg/ml BSA and the four dNTPs (50 μM).…”

Section: Methodsmentioning

confidence: 99%

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

Plaza-G.A.

¹

,

Lemishko

²

,

Crespo

³

et al. 2023

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…We used a miniaturized counter propagating dual-beam optical tweezers instrument (61) to manipulate individual DNA hairpins tethered between a streptavidin-coated bead (2.1 μm, Kisker Biotech) immobilized on top of a micropipette and an anti-digoxigenin-coated bead (3.0 μm diameter, Kisker Biotech) held in the optical trap, Figure 1B (62). Proteins were introduced inside the flow cell after dilution in the replication buffer containing 50 mM Tris pH 8.5, 30 mM KCl, 10 mM DTT, 4 mM MgCl 2 , 0.2 mg/ml BSA and the four dNTPs (50 μM).…”

Section: Methodsmentioning

confidence: 99%

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

Plaza-G.A.

¹

,

Lemishko

²

,

Crespo

³

et al. 2022

Preprint

Self Cite

2

0

View full text Add to dashboard Cite

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.

show abstract

“…There are a lot of important findings based on in vitro single molecule studies so far. [8][9][10][11] DAN packaging is a process to wrap the DNA with structural proteins, such as histone proteins, in order to store them in the nucleus. [12] DNA packaging and nucleosome remodeling have been believed to be another mechanism for gene expression regulation, called epigenesis, and have been studied a lot.…”

Section: Introductionmentioning

confidence: 99%

Single‐molecule tethered particle motion to study protein‐DNA interaction

Fan

¹

2023

J Chinese Chemical Soc

0

View full text Add to dashboard Cite

Single‐molecule techniques have been demonstrated as powerful tools to investigate individual protein‐DNA interactions that are difficult to access by conventional biochemical techniques. These methods are popularly used to obtain valuable and detailed molecular mechanisms of enzyme functions. Currently, we have used single‐molecule tethered particle motion to investigate protein‐DNA interactions, including homologous recombination, site‐specific recombination, DNA package, and the nucleosome remodeling process, and useful information was obtained. Here, we will describe the experimental designs and present the information obtained.

show abstract

DNA replication machinery: Insights from in vitro single-molecule approaches

Abstract: Graphical abstract

Cited by 7 publications

References 177 publications

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

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

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

Single‐molecule tethered particle motion to study protein‐DNA interaction

Contact Info

Product

Resources

About