Nonspecific DNA-protein Interaction Research Articles

Nonspecific Interaction between DNA and Protein allows for Cooperativity: A Case Study with Mycobacterium DNA Binding Protein

Different DNA-binding proteins have different interaction modes with DNA. Sequence-specific DNA-protein interaction has been mostly associated with regulatory processes inside a cell, and as such extensive studies have been made. Adequate data is also available on nonspecific DNA-protein interaction, as an intermediate to protein's search for its cognate partner. Multidomain nonspecific DNA-protein interaction involving physical sequestering of DNA has often been implicated to regulate gene expression indirectly. However, data available on this type of interaction is limited. One such interaction is the binding of DNA with mycobacterium DNA binding proteins. We have used the Langmuir-Blodgett technique to evaluate for the first time the kinetics and thermodynamics of Mycobacterium smegmatis Dps1 binding to DNA. By immobilizing one of the interacting partners, we have shown that, when a kinetic bottleneck is applied, the binding mechanism showed cooperative binding (n = 2.72) at lower temperatures, but the degree of cooperativity gradually reduces (n = 1.38) as the temperature was increased. We have also compared the kinetics and thermodynamics of sequence-specific and nonspecific DNA-protein interactions under the same set of conditions.

A kinetic Trapping Method for Evaluating One and Three Dimensional Target Site Location Mechanisms of Human Uracil DNA Glycosylase (UNG)

Maintaining the integrity of the encoded information within the human genome is a significant challenge for DNA repair systems. One such system is uracil base excision repair (BER) which begins by the action of Uracil DNA Glycosylase (UNG) that functions to remove incorporated uracils within the genome. While the enzymatic mechanisms and structures of BER glycosylase enzymes including UNG have been extensively studied, the diffusional mechanism by which these enzymes locate their target lesions efficiently among the high background of normal DNA is not well understood. A strategy that has been proposed is the use of facilitated transfer of the enzyme along DNA by non-specific DNA-protein interactions, which serves to increase the speed of target site location by reducing the search dimensionality from three to one dimension. While UNG and other glycosylases are considered to employ this strategy, the exact details are largely unknown. Here we describe a novel approach to unravel the short range DNA transfer mechanism of UNG using small molecule inhibitors that kinetically trap transient dissociated intermediates along the transfer pathway. We find that there is a very high probability that an UNG molecule will dissociate from DNA in the process of transferring between two uracil sites separated by only 20 bp along DNA. We use this kinetic trapping approach to estimate the net rate of intermolecular transfer between uracil sites as a function of site spacing, providing the first quantitative ensemble measurements of site transfer rates of a DNA repair enzyme.

Dynamical model of DNA-protein interaction: Effect of protein charge distribution and mechanical properties

The mechanical model based on beads and springs, which we recently proposed to study nonspecific DNA-protein interactions [J. Chem. Phys. 130, 015103 (2009)], was improved by describing proteins as sets of interconnected beads instead of single beads. In this paper, we first compare the results obtained with the updated model with those of the original one and then use it to investigate several aspects of the dynamics of DNA sampling, which could not be accounted for by the original model. These aspects include the effect on the speed of DNA sampling of the regularity and/or randomness of the protein charge distribution, the charge and location of the search site, and the shape and deformability of the protein. We also discuss the efficiency of facilitated diffusion, that is, the extent to which the combination of 1D sliding along the DNA and 3D diffusion in the cell can lead to faster sampling than pure 3D diffusion of the protein.

Nonspecific DNA-Protein Interaction: Why Proteins Can Diffuse along DNA

Recent single molecule experiments have reported that DNA binding proteins (DNA-BPs) can diffuse along DNA. This suggests that interactions between proteins and DNA play a role during the target search even far from their specific site on DNA. Here we show by means of Monte Carlo simulations and analytical calculations that there is a counterintuitive repulsion between the two oppositely charged macromolecules at a nanometer range. For the concave shape of DNA-BPs, and for realistic protein charge densities, we find that the DNA-protein interaction free energy has a minimum at a finite surface-to-surface separation, in which proteins can easily slide. When a protein encounters its target, the free energy barrier is completely counterbalanced by the H-bond interaction, thus enabling the sequence recognition.

From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Cα deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.

In Silico Study Of Nonspecific DNA-protein Encounter Complexes

In search of its specific targets, a DNA-binding protein associates with nonspecific DNA and subsequently diffuses along the DNA. Structural characterization of the nonspecific DNA-protein encounter complexes is of great interest. Due to weak interactions between the protein and nonspecific DNA, however, such characterization is experimentally challenging. Here, we describe the first comprehensive computational study on the encounter complexes of 44 specific DNA-binding proteins with nonspecific canonical B-DNA. In the analysis of these encounter complex models, we found that the recognition sites for specific DNA are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA-protein interaction modes exhibit some similarity to specific DNA-protein binding modes. These results led us to a novel method that predicts DNA-binding sites and binding-modes for a DNA-binding protein without knowing its specific DNA target sequence. In benchmark tests, the method achieves significantly better performance than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models up to 5 A from their native structures.

Description of nonspecific DNA-protein interaction and facilitated diffusion with a dynamical model

We propose a dynamical model for nonspecific DNA-protein interaction, which is based on the "bead-spring" model previously developed by other groups, and investigate its properties using Brownian dynamics simulations. We show that the model successfully reproduces some of the observed properties of real systems and predictions of kinetic models. For example, sampling of the DNA sequence by the protein proceeds via a succession of three-dimensional motion in the solvent, one-dimensional sliding along the sequence, short hops between neighboring sites, and intersegmental transfers. Moreover, facilitated diffusion takes place in a certain range of values of the protein effective charge, that is, the combination of one-dimensional sliding and three-dimensional motion leads to faster DNA sampling than pure three-dimensional motion. At last, the number of base pairs visited during a sliding event is comparable to the values deduced from single-molecule experiments. We also point out and discuss some discrepancies between the predictions of this model and some recent experimental results as well as some hypotheses and predictions of kinetic models.

Functional characterization of domains in AtTRB1, a putative telomere-binding protein in Arabidopsis thaliana

Telomeres are nucleoprotein structures ensuring the stability of eukaryotic chromosome ends. Two protein families, TRFL ( TFL- Like) and SMH ( Single- Myb- Histone), containing a specific telobox motif in their Myb domain, have been identified as potential candidates involved in a functional nucleoprotein structure analogous to human “shelterin” at plant telomeres. We analyze the DNA-protein interaction of the full-length and truncated variants of AtTRB1, a SMH-family member with a typical structure: N-terminal Myb domain, central H1/5 domain and C-terminal coiled-coil. We show that preferential interaction of AtTRB1 with double-stranded telomeric DNA is mediated by the Myb domain, while the H1/5 domain is involved in non-specific DNA-protein interaction and in the multimerization of AtTRB1.

Estimation of Förster's distance between two ends of Dps protein from mycobacteria: Distance heterogeneity as a function of oligomerization and DNA binding

Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.

Dynamics of Bacteriophage T4 Presynaptic Filament Assembly from Extrinsic Fluorescence Measurements of Gp32-Single-stranded DNA Interactions

In the bacteriophage T4 homologous recombination system, presynaptic filament assembly on single-stranded (ssDNA) DNA requires UvsX recombinase, UvsY mediator, and Gp32 ssDNA-binding proteins. Gp32 exerts both positive and negative effects on filament assembly: positive by denaturing ssDNA secondary structure, and negative by competing with UvsX for ssDNA binding sites. UvsY is believed to help UvsX displace Gp32 from the ssDNA. To test this model we developed a real-time fluorescence assay for Gp32-ssDNA interactions during presynapsis, based on changes in the fluorescence of a 6-iodoacetamidofluorescein-Gp32 conjugate. Results demonstrate that the formation of UvsX presynaptic filaments progressively disrupts Gp32-ssDNA interactions. Under stringent salt conditions the disruption of Gp32-ssDNA by UvsX is both ATP- and UvsY-dependent. The displacement of Gp32 from ssDNA during presynapsis requires ATP binding, but not ATP hydrolysis, by UvsX protein. Likewise, UvsY-mediated presynapsis strongly requires UvsY-ssDNA interactions, and is optimal at a 1:1 stoichiometry of UvsY to UvsX and/or ssDNA binding sites. Presynaptic filaments formed in the presence of UvsY undergo assembly/collapse that is tightly coupled to the ATP hydrolytic cycle and to stringent competition for ssDNA binding sites between Gp32 and various nucleotide-liganded forms of UvsX. The data directly support the Gp32 displacement model of UvsY-mediated presynaptic filament assembly, and demonstrate that the transient induction of high affinity UvsX-ssDNA interactions by ATP are essential, although not sufficient, for Gp32 displacement. The underlying dynamics of protein-ssDNA interactions within presynaptic filaments suggests that rearrangements of UvsX, UvsY, and Gp32 proteins on ssDNA may be coupled to central processes in T4 recombination metabolism.

How do site-specific DNA-binding proteins find their targets?

Essentially all the biological functions of DNA depend on site-specific DNA-binding proteins finding their targets, and therefore 'searching' through megabases of non-target DNA. In this article, we review current understanding of how this sequence searching is done. We review how simple diffusion through solution may be unable to account for the rapid rates of association observed in experiments on some model systems, primarily the Lac repressor. We then present a simplified version of the 'facilitated diffusion' model of Berg, Winter and von Hippel, showing how non-specific DNA-protein interactions may account for accelerated targeting, by permitting the protein to sample many binding sites per DNA encounter. We discuss the 1-dimensional 'sliding' motion of protein along non-specific DNA, often proposed to be the mechanism of this multiple site sampling, and we discuss the role of short-range diffusive 'hopping' motions. We then derive the optimal range of sliding for a few physical situations, including simple models of chromosomes in vivo, showing that a sliding range of approximately 100 bp before dissociation optimizes targeting in vivo. Going beyond first-order binding kinetics, we discuss how processivity, the interaction of a protein with two or more targets on the same DNA, can reveal the extent of sliding and we review recent experiments studying processivity using the restriction enzyme EcoRV. Finally, we discuss how single molecule techniques might be used to study the dynamics of DNA site-specific targeting of proteins.

Silencing Mediator for Retinoid and Thyroid Hormone Receptors Interacts with Octamer Transcription Factor-1 and Acts as a Transcriptional Repressor

Octamer transcription factor-1 (Oct-1) is a member of the POU (Pit-1, Oct-1, unc-86) family of transcription factors and is involved in the transcriptional regulation of a variety of gene expressions related to cell cycle regulation, development, and hormonal signals. It has been shown that Oct-1 acts not only as a transcriptional activator but also as a transcriptional repressor for certain genes. The mechanism of the repressive function of Oct-1 has not been well understood. Here we demonstrate by using the glutathione S-transferase pull-down assays and coimmunoprecipitation assays that the POU domain of Oct-1 directly interacts with a silencing mediator for retinoid and thyroid hormone receptors (SMRT). The interaction surfaces are located in the C-terminal region of SMRT, which are different from previously described silencing domains I and II or receptor interacting domains I and II. In transient transfection assays in COS1 cells, overexpression of SMRT attenuated the augmentation of Oct-1 transcriptional activity by OBF-1/OCA-B, activator for Oct-1. In pull-down assays, increasing amounts of SMRT could compete the binding of OCA-B to Oct-1 POU domain. The activity of Oct-1 could be determined by a regulated balance between SMRT and OCA-B. Furthermore, cotransfected unliganded thyroid hormone receptor enhanced the transactivation by Oct-1, and addition of 3,3',5-tri-iodo-l-thyronine obliterated the stimulatory effects. Consequently, in the presence of cotransfected thyroid hormone receptor, the octamer response element acts as an element negatively regulated by 3,3',5-tri-iodo-l-thyronine. The results suggest that the transcriptional activity of Oct-1 can be modulated by interaction through its POU domain by a silencing mediator SMRT resulting in the cross-talk between Oct-1 and nuclear receptors.

DNA Annealing and DNA–Protein Interactions by Capillary Electrophoresis

This work deals with annealing of single-stranded DNA and the binding of a serum respond factor to a DNA probe containing specific binding site. Capillary electrophoresis (CE) method is explored and compared with the mobility-shift gel electrophoresis (GE) procedure. The results indicate the CE method offers direct and rapid annealing of the DNA strands. It requires no prior incubation with additives (polynucleotides, proteins) to reduce nonspecific DNA–protein interactions. Unwanted nonspecific interactions are not observed in the CE method. The presence of a fluorescein tag to the DNA probe yields identical results to those with the radioactive label. A fluorescein tag in the CE work can be used without any adverse effects. The dissociation constant (Kd) of this protein–DNA complex by the CE method was similar to those determined by the GE method (≅10−6 M). The proposed method is extremely powerful, highly sensitive, quantitative, and fast. It can determine even very small conformational differences of the DNA probe.

Kinetics of RNA polymerase-promoter complex formation: effects of nonspecific DNA-protein interactions.

The rates of formation of RNA polymerase-promoter open complexes at the galactose P2 and lactose UV5 promoters of E. coli were studied using polyacrylamide gels to separate the heparin-resistant complexes from unbound DNA. Both the apparent rate and extent of reaction at these promoters are inhibited at excess RNA polymerase. This inhibition, which can be relieved by the addition of non-promoter DNA, is interpreted to be the result of occlusion of the promoter site by nonspecifically bound polymerase. Additionally, biphasic kinetics are observed at both gal P2 and lac UV5, but not at the PR promoter of phage lambda. This behavior disappears when the concentration of RNA polymerase in the binding reaction is less than that of the promoter fragment. It is proposed that at excess enzyme nonspecifically bound polymerase molecules sliding along the DNA may "bump" closed complexes from the promoter site thereby reducing the rate of open complex formation. Kinetics mechanisms quantifying both the occlusion and bumping phenomena are presented.

Free-sliding ligands: an alternative model of DNA-protein interactions.

AbstractWe present an alternative to the common lattice model for nonspecific DNA–protein interactions by using ligands that translate freely along the polynucleotide instead of binding to distinct lattice sites along the polynucleotide chain. The general model we present corresponds to a one‐dimensional continuum gas and is referred to as the “continuum model” to distinguish it from the general lattice model. Explicit expressions are obtained for the binding isotherm equation for two version of the continuum model, including the effects of binding‐site exclusion and attractions between bound ligands. Theoretical results are compared to those obtained from the McGhee‐von Hippel (1974) analysis of the lattice model with cooperative interactions between ligands occupying more than one lattice site. Practical applications of the continuum model are illustrated by analyzing (i) the noncooperative binding to single‐stranded DNA by RNase (Jensen and von Hippel, 1976), and (ii) the highly cooperative binding to poly(rA) by a proteolyzed fragment of the gene 32 protein of phage T4 (Lonberg et al., 1981).