How do proteins locate specific targets in DNA?

Redding, Sy; Greene, Eric C.

doi:10.1016/j.cplett.2013.03.035

Cited by 47 publications

(47 citation statements)

References 88 publications

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“…S2) suggests that the off-rate of Fis (and potentially other nucleoid proteins) might be accelerated in vivo beyond the level due to Fis-Fis exchange alone. The abundant nucleoid proteins, whose cellular levels vary with growth conditions (19), may therefore strongly influence dynamics of protein-DNA interactions on chromosomes, including protein-DNA search rates (1,33,34). Shifts in nucleoid protein concentration could provide a general mechanism to trigger rapid rearrangement of transcription factors and therefore reprogramming of transcriptional regulation.…”

Section: Facilitated Dissociation In Vitro Ex Vivo and In Vivomentioning

confidence: 99%

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 Vivomentioning

confidence: 99%

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

2016

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“…The nature of diffusive transport of DNA binding proteins in the context of target search has been of intense interest for decades and has extensive implications in many different facets of essential cellular processes, ranging from DNA replication and gene regulation to maintenance of genome stability (Kad et al, 2010; Lee et al, 2014a; Redding and Greene, 2013; Tafvizi et al, 2011b). The importance of search dimensionality was first pointed out by Adam and Delbruck, who suggested that the search process can be accelerated by collapsing a three-dimensional search into a one-dimensional search along the DNA (Adam and Delbrück, 1968).…”

Section: The Target Search Problem: Solving the Speed-stability Pamentioning

confidence: 99%

Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins

Kong

Houten

2017

Progress in Biophysics and Molecular Biology

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Since Robert Brown’s first observations of random walks by pollen particles suspended in solution, the concept of diffusion has been subject to countless theoretical and experimental studies in diverse fields from finance and social sciences, to physics and biology. Diffusive transport of macromolecules in cells is intimately linked to essential cellular functions including nutrient uptake, signal transduction, gene expression, as well as DNA replication and repair. Advancement in experimental techniques has allowed precise measurements of these diffusion processes. Mathematical and physical descriptions and computer simulations have been applied to model complicated biological systems in which anomalous diffusion, in addition to simple Brownian motion, was observed. The purpose of this review is to provide an overview of the major physical models of anomalous diffusion and corresponding experimental evidence on the target search problem faced by DNA-binding proteins, with an emphasis on DNA repair proteins and the role of anomalous diffusion in DNA target recognition.

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“…Similarly, 55% of MutLα found mismatch-bound MutSα through pathway involving a 1D search, whereas the remaining 45% used a 3D association pathway. This ability to engage lesions through either 1D or 3D pathways is not surprising or even unexpected; moreover it is crucial to recognize that the ratio of 1D to 3D binding events is expected to change as a function of protein concentration [37, 38]. The fraction of proteins utilizing a 1D search will always increase at low protein concentrations, whereas the fraction of those that bind through a 3D pathway will always increase at higher protein concentrations [37, 38].…”

Section: Mmr Proteins As Models For Studying Diffusive Motion On Dnamentioning

confidence: 99%

“…This ability to engage lesions through either 1D or 3D pathways is not surprising or even unexpected; moreover it is crucial to recognize that the ratio of 1D to 3D binding events is expected to change as a function of protein concentration [37, 38]. The fraction of proteins utilizing a 1D search will always increase at low protein concentrations, whereas the fraction of those that bind through a 3D pathway will always increase at higher protein concentrations [37, 38]. This concentration-dependent effect on target searches needs to be considered when trying to interpret how results obtained from a single-molecule measurement of 1D diffusion might pertain to in vivo scenarios where the protein concentrations (among other things; see below) might differ substantially from in vitro conditions.…”

Section: Mmr Proteins As Models For Studying Diffusive Motion On Dnamentioning

confidence: 99%

“…There are two potential mechanisms that might allow MutSα to conduct effect target searches on DNA bound by nucleosomes: either something must remove the nucleosomes from the DNA, and/or the high in vivo concentration of MutS may obviate the need for 1D sliding during the target search by driving the preference for mismatch binding through a 3D pathway [37, 38]. Recent work by Hombauer and colleagues suggest that both possibilities may MMR in vivo [48].…”

Section: Mmr Proteins As Models For Studying Diffusive Motion On Dnamentioning

confidence: 99%

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Visualizing protein movement on DNA at the single-molecule level using DNA curtains

2014

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A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively.

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How do proteins locate specific targets in DNA?

Cited by 47 publications

References 88 publications

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins

Visualizing protein movement on DNA at the single-molecule level using DNA curtains

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