From “Simple” DNA-Protein Interactions to the Macromolecular Machines of Gene Expression

Hippel, Peter H. von

doi:10.1146/annurev.biophys.34.040204.144521

Cited by 180 publications

(154 citation statements)

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“…First, nonconsensus protein−DNA binding might provide an explanation for the so-called highly occupied target regions, which are depleted in known consensus motifs yet are highly enriched in repeated DNA sequences with unknown function (18,33). Second, nonconsensus sequence elements flanking specific consensus motifs are likely to significantly affect the properties of the kinetic search that a protein performs to find its specific target sites on DNA (7)(8)(9)(10)34). Our results demonstrate that nonconsensus DNA background exerts substantially nonrandom statistical potential on DNA-binding proteins, and therefore, such potential must be taken into account to predict the kinetics of protein−DNA binding.…”

Section: Discussionmentioning

confidence: 99%

“…protein−DNA binding is an important biophysical mechanism operating in a living cell (1). This seminal work makes it possible to interpret experiments that measured how transcription factors (TFs) search for their specific target sites flanked by nonconsensus sequence elements (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). A specific consensus motif is a short DNA sequence, typically 6-20 base pairs (bp), that possesses an enhanced binding affinity for a particular TF.…”

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confidence: 99%

“…1). The process of establishing specific, consensus protein−DNA binding requires the formation of precise geometrical fit between the protein and its consensus DNA motif, accompanied by the formation of specific hydrogen and electrostatic contacts at the protein−DNA binding interface (6,7) (Fig. 1).…”

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confidence: 99%

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Protein−DNA binding in the absence of specific base-pair recognition

Afek

Schipper

Horton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

105

134

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Until now, it has been reasonably assumed that specific base-pair recognition is the only mechanism controlling the specificity of transcription factor (TF)−DNA binding. Contrary to this assumption, here we show that nonspecific DNA sequences possessing certain repeat symmetries, when present outside of specific TF binding sites (TFBSs), statistically control TF−DNA binding preferences. We used highthroughput protein−DNA binding assays to measure the binding levels and free energies of binding for several human TFs to tens of thousands of short DNA sequences with varying repeat symmetries. Based on statistical mechanics modeling, we identify a new protein−DNA binding mechanism induced by DNA sequence symmetry in the absence of specific base-pair recognition, and experimentally demonstrate that this mechanism indeed governs protein−DNA binding preferences. protein−DNA binding is an important biophysical mechanism operating in a living cell (1). This seminal work makes it possible to interpret experiments that measured how transcription factors (TFs) search for their specific target sites flanked by nonconsensus sequence elements (1-10). A specific consensus motif is a short DNA sequence, typically 6-20 base pairs (bp), that possesses an enhanced binding affinity for a particular TF. For example, the sequence CACGTG represents the specific consensus motif for the human protein Max used in this study (Fig. 1). The process of establishing specific, consensus protein−DNA binding requires the formation of precise geometrical fit between the protein and its consensus DNA motif, accompanied by the formation of specific hydrogen and electrostatic contacts at the protein−DNA binding interface (6, 7) ( Fig. 1). In addition to binding to their consensus DNA motifs, transcription factors can also bind, albeit with lower affinity, to DNA regions lacking any consensus motifs. The term "nonspecific protein−DNA binding" (6) is typically used to describe these weaker interactions. Von Hippel and Berg suggested classifying nonspecific protein−DNA binding into two related mechanisms (6). The first mechanism includes protein binding to its mutated specific motifs that retain some residual, reduced specificity. The second mechanism is largely DNA sequence independent, and it involves electrostatic binding modulated by the overall DNA geometry (6). Despite significant experimental progress, molecular mechanisms responsible for these two types of nonspecific binding remain poorly understood, and the free energy of nonspecific protein−DNA binding has not been systematically characterized (11)(12)(13)(14). The interplay between consensus and nonconsensus DNA sequence elements emerges as a dominant factor that governs protein−DNA binding preferences. However, this interplay is also poorly understood (15, 16). Until now, it has been reasonably assumed that specific (consensus) base-pair recognition must control the genome-wide specificity of TF−DNA binding.Contrary to this assumption, here we identify a general mechanism for protein−DNA bi...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Protein−DNA binding in the absence of specific base-pair recognition

Afek

Schipper

Horton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

105

134

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“…Second, an alternate set of buffer conditions was tested. Studies of the cooperative binding properties of single-strand binding (Ssb) protein, for example, indicate that the binding behavior of single-strand binding protein can be greatly altered by the salt and divalent metal ion concen- tration in the reaction (33). In addition, previous experiments suggesting that Rap1 may bend DNA were performed under conditions different from above.…”

Section: Rap1 Is a Monomer With A Ringlikementioning

confidence: 99%

Characterization of the Yeast Telomere Nucleoprotein Core

Williams

Levy

Maki-Yonekura³

et al. 2010

Journal of Biological Chemistry

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At the core of Saccharomyces cerevisiae telomeres is an array of tandem telomeric DNA repeats bound site-specifically by multiple Rap1 molecules. There, Rap1 orchestrates the binding of additional telomere-associated proteins and negatively regulates both telomere fusion and length homeostasis. Using electron microscopy, viscosity, and light scattering measurements, we show that purified Rap1 is a monomer in solution that adopts a ringlike or C shape with a central cavity. Rap1 could orchestrate telomere function by binding multiple telomere array sites through either cooperative or independent mechanisms. To determine the mechanism, we analyze the distribution of Rap1 monomers on defined telomeric DNA arrays. This analysis clearly indicates that Rap1 binds independently to each nonoverlapping site in an array, regardless of the spacing between sites, the total number of sites, the affinity of the sites for Rap1, and over a large concentration range. Previous experiments have not clearly separated the effects of affinity from repeat spacing on telomere function. We clarify these results by testing in vivo the function of defined telomere arrays containing the same Rap1 binding site separated by spacings that were previously defined as low or high activity. We find that Rap1 binding affinity in vitro correlates with the ability of telomeric repeat arrays to regulate telomere length in vivo. We suggest that Rap1 binding to multiple sites in a telomere array does not, by itself, promote formation of a more energetically stabile complex.

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“…Thus, protein binding is tighter at contiguous sites because of the additional interactions between bound proteins (Figure 1, panel b). Binding to a contiguous site may contribute up to 3Ϫ4 kcal mol Ϫ1 of more favorable binding free energy (12).…”

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confidence: 99%