DNA conformation and protein DNA interaction

Issued as Preliminary report, Project no. G-41-B05 (continues G-41-B04; continued by G-41-B06

Kinetics of protein-DNA interaction: facilitated target location in sequence-dependent potential

Recognition and binding of specific sites on DNA by proteins is central for many cellular functions such as transcription, replication, and recombination. In the process of recognition, a protein rapidly searches for its specific site on a long DNA molecule and then strongly binds this site. Here we aim to find a mechanism that can provide both a fast search (1-10 sec) and high stability of the specific protein-DNA complex ($K_d=10^{-15}-10^{-8}$ M). Earlier studies have suggested that rapid search involves the sliding of a protein along the DNA. Here we consider sliding as a one-dimensional (1D) diffusion in a sequence-dependent rough energy landscape. We demonstrate that, in spite of the landscape's roughness, rapid search can be achieved if 1D sliding is accompanied by 3D diffusion. We estimate the range of the specific and non-specific DNA-binding energy required for rapid search and suggest experiments that can test our mechanism. We show that optimal search requires a protein to spend half of time sliding along the DNA and half diffusing in 3D. We also establish that, paradoxically, realistic energy functions cannot provide both rapid search and strong binding of a rigid protein. To reconcile these two fundamental requirements we propose a search-and-fold mechanism that involves the coupling of protein binding and partial protein folding. Proposed mechanism has several important biological implications for search in the presence of other proteins and nucleosomes, simultaneous search by several proteins etc. Proposed mechanism also provides a new framework for interpretation of experimental and structural data on protein-DNA interactions

ChIPOTle: a user-friendly tool for the analysis of ChIP-chip data

ChIPOTle (Chromatin ImmunoPrecipitation On Tiled arrays) takes advantage of two unique properties of ChIP-chip data: the single-tailed nature of the data, caused by specific enrichment but not specific depletion of genomic fragments; and the predictable enrichment of DNA fragments adjacent to sites of direct protein-DNA interaction. Implemented as a Microsoft Excel macro written in Visual Basic, ChIPOTle uses a sliding window approach that yields improvements in the identification of bona fide sites of protein-DNA interaction

Rational design of DNA sequence-specific zinc fingers

AbstractWe developed a rational scheme for designing DNA binding proteins. The scheme was applied for a zinc finger protein and the designed sequences were experimentally characterized with high DNA sequence specificity. Starting with the backbone of a known finger structure, we initially calculated amino acid sequences compatible with the expected structure and the secondary structures of the designed fingers were then experimentally confirmed. The DNA-binding function was added to the designed finger by reconsidering a section of the amino acid sequence and computationally selecting amino acids to have the lowest protein–DNA interaction energy for the target DNA sequences. Among the designed proteins, one had a gap between the lowest and second lowest protein–DNA interaction energies that was sufficient to give DNA sequence-specificity

Predicting variation of DNA shape preferences in protein-DNA interaction in cancer cells with a new biophysical model

DNA shape readout is an important mechanism of target site recognition by transcription factors, in addition to the sequence readout. Several models of transcription factor-DNA binding which consider DNA shape have been developed in recent years. We present a new biophysical model of protein-DNA interaction by considering the DNA shape features, which is based on a neighbour dinucleotide dependency model BayesPI2. The parameters of the new model are restricted to a subspace spanned by the 2-mer DNA shape features, which allowing a biophysical interpretation of the new parameters as position-dependent preferences towards certain values of the features. Using the new model, we explore the variation of DNA shape preferences in several transcription factors across cancer cell lines and cellular conditions. We find evidence of DNA shape variations at FOXA1 binding sites in MCF7 cells after treatment with steroids. The new model is useful for elucidating finer details of transcription factor-DNA interaction. It may be used to improve the prediction of cancer mutation effects in the future

Fluorescent microplate-based analysis of protein-DNA interactions I:immobilized protein

A simple protein-DNA interaction analysis has been developed using a high-affinity/high-specificity zinc finger protein. In essence, purified protein samples are immobilized directly onto the surface of microplate wells, and fluorescently labeled DNA is added in solution. After incubation and washing, bound DNA is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.2 nM DNA. Since the detection of bound DNA is noninvasive and the protein-DNA interaction is not disrupted during detection, iterative readings may be taken from the same well, after successive alterations in interaction conditions, if required. In this respect, the assay may therefore be considered real time and permits appropriate interaction conditions to be determined quantitatively. The assay format is ideally suited to investigate the interactions of purified unlabeled DNA binding proteins in a high-throughput format

Design of a combinatorial DNA microarray for protein-DNA interaction studies

BACKGROUND: Discovery of precise specificity of transcription factors is an important step on the way to understanding the complex mechanisms of gene regulation in eukaryotes. Recently, double-stranded protein-binding microarrays were developed as a potentially scalable approach to tackle transcription factor binding site identification. RESULTS: Here we present an algorithmic approach to experimental design of a microarray that allows for testing full specificity of a transcription factor binding to all possible DNA binding sites of a given length, with optimally efficient use of the array. This design is universal, works for any factor that binds a sequence motif and is not species-specific. Furthermore, simulation results show that data produced with the designed arrays is easier to analyze and would result in more precise identification of binding sites. CONCLUSION: In this study, we present a design of a double stranded DNA microarray for protein-DNA interaction studies and show that our algorithm allows optimally efficient use of the arrays for this purpose. We believe such a design will prove useful for transcription factor binding site identification and other biological problems

Base pair opening and bubble transport in a DNA double helix induced by a protein molecule in a viscous medium

We study the nonlinear dynamics of a protein-DNA molecular system by treating DNA as a set of two coupled linear chains and protein in the form of a single linear chain sliding along the DNA at the physiological temperature in a viscous medium. The nonlinear dynamics of the above molecular system in general is governed by a perturbed nonlinear Schr\"{o}dinger equation. In the non-viscous limit, the equation reduces to the completely integrable nonlinear Schr\"{o}dinger (NLS) equation which admits N-soliton solutions. The soliton excitations of the DNA bases make localized base pair opening and travel along the DNA chain in the form of a bubble. This may represent the bubble generated during the transcription process when an RNA-polymerase binds to a promoter site in the DNA double helical chain. The perturbed NLS equation is solved using a perturbation theory by treating the viscous effect due to surrounding as a weak perturbation and the results show that the viscosity of the solvent in the surrounding damps out the amplitude of the soliton.Comment: 4. Submitted to Phys. Rev.

The long reach of DNA sequence heterogeneity in diffusive processes

Many biological processes involve one dimensional diffusion over a correlated inhomogeneous energy landscape with a correlation length $\xi_c$. Typical examples are specific protein target location on DNA, nucleosome repositioning, or DNA translocation through a nanopore, in all cases with $\xi_c\approx$ 10 nm. We investigate such transport processes by the mean first passage time (MFPT) formalism, and find diffusion times which exhibit strong sample to sample fluctuations. For a a displacement $N$, the average MFPT is diffusive, while its standard deviation over the ensemble of energy profiles scales as $N^{3/2}$ with a large prefactor. Fluctuations are thus dominant for displacements smaller than a characteristic $N_c \gg \xi_c$: typical values are much less than the mean, and governed by an anomalous diffusion rule. Potential biological consequences of such random walks, composed of rapid scans in the vicinity of favorable energy valleys and occasional jumps to further valleys, is discussed