Stochastic Ratchet Mechanisms for Replacement of Proteins Bound to DNA

Cocco, Simona; Marko, John F.; Monasson, Rémi

doi:10.1103/physrevlett.112.238101

Cited by 28 publications

(38 citation statements)

References 14 publications

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“…This facilitated dissociation effect had been observed previously in vitro on extended -DNA molecules (10,32). The experiments in this study establish that a similar effect with remarkably similar kinetic rate (k exch Ϸ 10 4 M Ϫ1 s Ϫ1 for 100 mM NaCl) occurs for folded chromosomes isolated from E. coli cells.…”

Section: Facilitated Dissociation In Vitro Ex Vivo and In Vivosupporting

confidence: 64%

“…The slope of the linear regime can be interpreted as an exchange reaction rate constant (k exch ) (26) Decreased salt concentration reduces the rate of facilitated dissociation. A possible physical origin of the exchange reactions observed by Graham et al has been proposed to be "microdissociation" events, whereby a protein loses some or all of its interactions with its binding site but remains near the DNA due to partial binding (11) or perhaps to longer-ranged electrostatic interactions (10,32). In the absence of solution-phase protein, it is likely that the partially dissociated protein rebinds to the DNA.…”

Section: Isolation and Visualization Of Nucleoidsmentioning

confidence: 99%

“…Of course, it may be that the ternary complex has an ϳ100-s lifetime preceding protein dissociation; this should be observable in a two-color single-molecule visualization experiment. While definitive data deciding which of these mechanisms (or perhaps some other mechanism) actually occurs may require millisecond/nanometer single-molecule molecular dynamics monitoring experiments, at present we favor models whereby an activation step of the Fis-DNA complex precedes interaction with the second invading protein (5,10,11).…”

Section: Facilitated Dissociation In Vitro Ex Vivo and In Vivomentioning

confidence: 99%

“…This indicates that the effect of the invader-i.e., its initial interaction with the prebound Fis-DNA complex-is rate limited not by diffusive search but rather by some other preceding reaction step, the activation of which has associated with it a probability of ϳ10 Ϫ4 (corresponding to an activation barrier of ϳ12 k B T, where k B T ϭ 4.1 ϫ 10 Ϫ21 J is the thermal energy per molecule at ambient temperature~300 K) (5). This is very naturally explained in terms of a partial dissociation or other molecular reorganization that makes the Fis-DNA complex susceptible to invasion by the solution Fis dimer (5,10,11) and is much less easily understood in terms of diffusion-limited binding of the second dimer followed by (presumably rapid [12]) allosteric interaction.…”

Section: Facilitated Dissociation In Vitro Ex Vivo and In Vivomentioning

confidence: 99%

“…A protein in bulk solution could impact dissociation events in a variety of ways (5,9). This interaction can affect the rate of macroscopic dissociation of the original protein, for example, by having the second "invading" protein "block" rebinding of the first protein (5,10,11). Alternatively, both the initially bound and invading proteins might bind at adjacent sites, with the second protein accelerating the off-rate of the first protein via allosteric interactions (12).…”

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Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

2016

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Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or "facilitated dissociation." Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an "exchange" rate constant (k exch ) of Ϸ10 4 M ؊1 s ؊1 , comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo. A ll aspects of chromosome dynamics involve the binding and unbinding of proteins from the DNA double helix. The rate of binding for a given species of protein to a location along a DNA is usually controlled by concentration of that species, but it is widely assumed that unbinding times, or equivalently off-rates, are controlled by the strength of interactions inside the DNA-protein complex and are independent of other nearby molecules in the nucleoplasm (1). However, this picture has been challenged by a number of recent in vitro experiments which have shown off-rates of proteins from duplex DNA that depend on bulk protein concentrations (2-5). Similar effects have been observed for singlestranded DNA (ssDNA)-binding proteins (6, 7), antibody-ligand binding (8), and ssDNA-ssDNA interactions (9).A straightforward explanation for this effect is that macroscopic dissociation (complete dissociation followed by diffusive motion of a protein to a location far away from the initial binding site) occurs via one or more partially dissociated intermediate ("microdissociated") states. A protein in bulk solution could impact dissociation events in a variety of ways (5, 9). This interaction can affect the rate of macroscopic dissociation of the original protein, for example, by having the second "invading" protein "block" rebinding of the first protein (5, 10, 11). Alternatively, both the initially bound and invading proteins might bind at adjacent sites, with the second protein accelerating the off-rate of the first protein via allosteric interactions (12). In either case, formation of a transient ternary complex (initially bound protein plus DNA site plus invading protein) may lead to appreciable increases in off-rates as the bulk protein concentration is increased. In the molecularly crowded cell interior, the rates of replacement of proteins on DNA may well be very different from what might be predicted from dilute-solution studies of binding kinetics, a fact that may help reconcile observations of rapid turnover on DNA in vivo but slower kinetics observed in vitro (13)(14)(15)(16)(17).An example o...

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Section: Facilitated Dissociation In Vitro Ex Vivo and In Vivosupporting

confidence: 64%

Section: Isolation and Visualization Of Nucleoidsmentioning

confidence: 99%

Section: Facilitated Dissociation In Vitro Ex Vivo and In Vivomentioning

confidence: 99%

Section: Facilitated Dissociation In Vitro Ex Vivo and In Vivomentioning

confidence: 99%

mentioning

confidence: 99%

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Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

2016

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Single-Stranded DNA Curtains for Studying Homologous Recombination

Steinfeld

Greene

2017

Methods in Enzymology

113

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Homologous recombination is an important pathway involved in the repair of double-stranded DNA breaks. Genetic studies form the foundation of our knowledge on homologous recombination. Significant progress has also been made toward understanding the biochemical and biophysical properties of the proteins, complexes, and reaction intermediates involved in this essential DNA repair pathway. However, heterogeneous or transient recombination intermediates remain extremely difficult to assess through traditional ensemble methods, leaving an incomplete mechanistic picture of many steps that take place during homologous recombination. To help overcome some of these limitations, we have established DNA curtain methodologies as an experimental platform for studying homologous DNA recombination in real-time at the single-molecule level. Here, we present a detailed overview describing the preparation and use of single-stranded DNA curtains in applications related to the study of homologous DNA recombination with emphasis on recent work related to the study of the eukaryotic recombinase Rad51.

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Facilitated Dissociation Kinetics of Dimeric Nucleoid-Associated Proteins Follow a Universal Curve

Dahlke

Sing

2017

Biophysical Journal

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Recent experimental work has demonstrated facilitated dissociation of certain nucleoid-associated proteins that exhibit an unbinding rate that depends on the concentration of freely diffusing proteins or DNA in solution. This concentration dependence arises due to binding competition with these other proteins or DNA. The identity of the binding competitor leads to different qualitative trends, motivating an investigation to understand observed differences in facilitated dissociation. We use a coarse-grained simulation that takes into account the dimeric nature of many nucleoid-associated proteins by allowing an intermediate binding state. The addition of this partially bound state allows the protein to be unbound, partially bound, or fully bound to a DNA strand, leaving opportunities for other molecules in solution to participate in the unbinding mechanism. Previous models postulated symmetric binding energies for each state of the coarse-grained protein corresponding to the symmetry of the dimeric protein; this model relaxes this assumption by assigning different energies for the different steps in the unbinding process. Allowing different unbinding energies not only has equilibrium effects on the system, but kinetic effects as well. We were able to reproduce the unbinding trends seen experimentally for both DNA and protein competitors. All trends collapse to a universal curve regardless of the unbinding energies used or the identity of the dissociation facilitator, suggesting that facilitated dissociation can be described with a single set of scaling parameters that are related to the energy landscape and geometric nature of the competitors.

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Stochastic Ratchet Mechanisms for Replacement of Proteins Bound to DNA

Cited by 28 publications

References 14 publications

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

Single-Stranded DNA Curtains for Studying Homologous Recombination

Facilitated Dissociation Kinetics of Dimeric Nucleoid-Associated Proteins Follow a Universal Curve

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