Investigation of Protein–Protein Interactions of Single-Stranded DNA-Binding Proteins by Analytical Ultracentrifugation

Naue, Natalie; Curth, Ute

doi:10.1007/978-1-62703-032-8_8

Cited by 8 publications

(9 citation statements)

References 25 publications

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“…The χ subunit of the Pol III HE interacts with the C-terminal tail of SSB and facilitates binding to and elongating templates that are coated with SSB. 21; 22; 23; 24 We have observed that an interaction between Pol III HE component other than χ and the C-terminal tail of SSB is required for the optimal efficiency of initiation complex formation under conditions where Pol III associated with τ-containing DnaX complexes is chaperoned onto newly assembled β 2 . 25 During initiation complex formation in the presence of single-tailed SSB-LT-Drl it is possible that a portion of the Pol III HE interacts through χ precluding stimulation by the second interaction site or even trapping the enzyme in a non-productive complex.…”

Section: Discussionmentioning

confidence: 99%

“…The DNA polymerase III holoenzyme (Pol III HE) consists of a DNA Pol III core (α-ε-θ), the multi-subunit DnaX complex clamp loader (τ, γ, δ, δ′, χ and ψ subunits) and the β clamp, a processivity factor. SSB binds to the χψ complex within the clamp loader 21; 22 and contributes to processive replication. 23; 24 A second interaction of SSB with a Pol III HE site, other than χ, contributes to rapid initiation complex formation in a process where the DnaX complex chaperones Pol III onto β 2 loaded in the same reaction cycle.…”

Section: Introductionmentioning

confidence: 99%

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Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Antony

Weiland

Yuan

et al. 2013

Journal of Molecular Biology

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E. coli single strand DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function we engineered covalently linked forms of SSB that possess only one or two C-termini within a four OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents, but accumulate more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Antony

Weiland

Yuan

et al. 2013

Journal of Molecular Biology

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“…Therefore, the reaction must be fast on the timescale of sedimentation (52). As previously described for the SSB/χ interaction (29,46,47,53), integration of the two peaks in the c ( s ) distribution can be used to determine the concentrations of free and bound SSB-binding partner and subsequently the binding parameters. Fitting a model of four identical independent binding sites for DnaG-C on an EcoSSB tetramer to the data revealed an association constant ( K A ) of ∼9 × 10 4 M − 1 (Figure 3).…”

Section: Resultsmentioning

confidence: 99%

“…This program provides a model for diffusion-deconvoluted differential sedimentation coefficient distributions [ c ( s ) distributions]. As the areas under the separate peaks in c ( s ) distributions are a measure of the absorbance of the species represented by the peaks (45), this information can be used to determine binding isotherms (29,46,47). …”

Section: Methodsmentioning

confidence: 99%

The helicase-binding domain of Escherichia coli DnaG primase interacts with the highly conserved C-terminal region of single-stranded DNA-binding protein

Naue

Beerbaum

Bogutzki

et al. 2013

Nucleic Acids Research

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During bacterial DNA replication, DnaG primase and the χ subunit of DNA polymerase III compete for binding to single-stranded DNA-binding protein (SSB), thus facilitating the switch between priming and elongation. SSB proteins play an essential role in DNA metabolism by protecting single-stranded DNA and by mediating several important protein–protein interactions. Although an interaction of SSB with primase has been previously reported, it was unclear which domains of the two proteins are involved. This study identifies the C-terminal helicase-binding domain of DnaG primase (DnaG-C) and the highly conserved C-terminal region of SSB as interaction sites. By ConSurf analysis, it can be shown that an array of conserved amino acids on DnaG-C forms a hydrophobic pocket surrounded by basic residues, reminiscent of known SSB-binding sites on other proteins. Using protein–protein cross-linking, site-directed mutagenesis, analytical ultracentrifugation and nuclear magnetic resonance spectroscopy, we demonstrate that these conserved amino acid residues are involved in the interaction with SSB. Even though the C-terminal domain of DnaG primase also participates in the interaction with DnaB helicase, the respective binding sites on the surface of DnaG-C do not overlap, as SSB binds to the N-terminal subdomain, whereas DnaB interacts with the ultimate C-terminus.

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“…After accounting for a characteristic signal structure imposed by the optical configuration (46), it offers highly quantitative representation of the sedimentation process (46,47), and allows the detection of low picomolar concentrations of common fluorophores (32,48). This has permitted studies of labeled proteins in complex fluids such as serum (49,50) and cell lysates (29,51), and diverse studies of high-affinity interacting systems (48,(52)(53)(54)(55) with K d as low as 20 pM (48).…”

Section: Introductionmentioning

confidence: 99%

Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield

Chaturvedi

Zhao

Schuck

2017

Biophysical Journal

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Sedimentation velocity analytical ultracentrifugation with fluorescence detection has emerged as a powerful method for the study of interacting systems of macromolecules. It combines picomolar sensitivity with high hydrodynamic resolution, and can be carried out with photoswitchable fluorophores for multicomponent discrimination, to determine the stoichiometry, affinity, and shape of macromolecular complexes with dissociation equilibrium constants from picomolar to micromolar. A popular approach for data interpretation is the determination of the binding affinity by isotherms of weight-average sedimentation coefficients s. A prevailing dogma in sedimentation analysis is that the weight-average sedimentation coefficient from the transport method corresponds to the signal- and population-weighted average of all species. We show that this does not always hold true for systems that exhibit significant signal changes with complex formation-properties that may be readily encountered in practice, e.g., from a change in fluorescence quantum yield. Coupled transport in the reaction boundary of rapidly reversible systems can make significant contributions to the observed migration in a way that cannot be accounted for in the standard population-based average. Effective particle theory provides a simple physical picture for the reaction-coupled migration process. On this basis, we develop a more general binding model that converges to the well-known form of s with constant signals, but can account simultaneously for hydrodynamic cotransport in the presence of changes in fluorescence quantum yield. We believe this will be useful when studying interacting systems exhibiting fluorescence quenching, enhancement, or Förster resonance energy transfer with transport methods.

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Investigation of Protein–Protein Interactions of Single-Stranded DNA-Binding Proteins by Analytical Ultracentrifugation

Cited by 8 publications

References 25 publications

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

The helicase-binding domain of Escherichia coli DnaG primase interacts with the highly conserved C-terminal region of single-stranded DNA-binding protein

Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield

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