The kinetic landscape of nucleosome assembly: A coarse-grained molecular dynamics study

The organization of nucleosomes along the Eukaryotic genome is maintained over time despite disruptive events such as replication. During this complex process, histones and DNA can form a variety of non-canonical nucleosome conformations, but their precise molecular details and roles during nucleosome assembly remain unclear. In this study, employing coarse-grained molecular dynamics simulations and Markov state modeling, we characterized the complete kinetics of nucleosome assembly. On the nucleosome-positioning 601 DNA sequence, we observe a rich transition network among various canonical and non-canonical tetrasome, hexasome, and nucleosome conformations. A low salt environment makes nucleosomes stable, but the kinetic landscape becomes more rugged, so that the system is more likely to be trapped in off-pathway partially assembled intermediates. Finally, we find that the co-operativity between DNA bending and histone association enables positioning sequence motifs to direct the assembly process, with potential implications for the dynamic organization of nucleosomes on real genomic sequences

Ensembles of breathing nucleosomes: a computational study

Theoretical Physic

Ensembles of Breathing Nucleosomes: A Computational Study

Theoretical PhysicsBiological and Soft Matter Physic

Understanding protein diffusion on force-induced stretched DNA conformation

DNA morphology is subjected to environmental conditions and is closely coupled with its function. For example, DNA experiences stretching forces during several biological processes, including transcription and genome transactions, that significantly alter its conformation from that of B-DNA. Indeed, a well-defined 1.5 times extended conformation of dsDNA, known as Σ-DNA, has been reported in DNA complexes with proteins such as Rad51 and RecA. A striking feature in Σ-DNA is that the nucleobases are partitioned into triplets of three locally stacked bases separated by an empty rise gap of ∼5 Å. The functional role of such a DNA base triplet was hypothesized to be coupled with the ease of recognition of DNA bases by DNA-binding proteins (DBPs) and the physical origin of three letters (codon/anti-codon) in the genetic code. However, the underlying mechanism of base-triplet formation and the ease of DNA base-pair recognition by DBPs remain elusive. To investigate, here, we study the diffusion of a protein on a force-induced stretched DNA using coarse-grained molecular dynamics simulations. Upon pulling at the 3′ end of DNA by constant forces, DNA exhibits a conformational transition from B-DNA to a ladder-like S-DNA conformation via Σ-DNA intermediate. The resulting stretched DNA conformations exhibit non-uniform base-pair clusters such as doublets, triplets, and quadruplets, of which triplets are energetically more stable than others. We find that protein favors the triplet formation compared to its unbound form while interacting non-specifically along DNA, and the relative population of it governs the ruggedness of the protein–DNA binding energy landscape and enhances the efficiency of DNA base recognition. Furthermore, we analyze the translocation mechanism of a DBP under different force regimes and underscore the significance of triplet formation in regulating the facilitated diffusion of protein on DNA. Our study, thus, provides a plausible framework for understanding the structure–function relationship between triplet formation and base recognition by a DBP and helps to understand gene regulation in complex regulatory processes

多分子及び一分子測定によるp53の標的探索ダイナミクスの解明

Tohoku University髙橋聡課

A Study of p53 Action on DNA at the Single Molecule Level

The transcription factor p53 searches for and binds to target sequences within long genomic DNA, to regulate downstream gene expression. p53 possesses multiple disordered and DNA-binding domains, which are frequently observed in DNA-binding proteins. Owing to these properties, p53 is used as a model protein for target search studies. It counters cell stress by utilizing a facilitated diffusion mechanism that combines 3D diffusion in solution, 1D sliding along DNA, hopping/jumping along DNA, and intersegmental transfer between two DNAs. Single-molecule fluorescence microscopy has been used to characterize individual motions of p53 in detail. In addition, a biophysical study has revealed that p53 forms liquid-like droplets involving the functional switch. In this chapter, the target search and regulation of p53 are discussed in terms of dynamic properties

Particle Filter Method to Integrate High-Speed Atomic Force Microscopy Measurements with Biomolecular Simulations

High-speed atomic force microscopy (HS-AFM) can be used to observe the structural dynamics of biomolecules at the single-molecule level in real time under near-physiological conditions; however, its spatiotemporal resolution is limited. Complementarily, molecular dynamics (MD) simulations have higher spatiotemporal resolutions, albeit with some artifacts. Here, to integrate HS-AFM data and coarse-grained molecular dynamics (CG-MD) simulations, we develop a particle filter method that implements a sequential Bayesian data assimilation approach. We test the method in a twin experiment. First, we generate a reference HS-AFM movie from the CG-MD trajectory of a test molecule, a nucleosome; this serves as the “experimental measurement”. Then, we perform a particle filter simulation with 512 particles, which captures the large-scale nucleosome structural dynamics compatible with the AFM movie. Comparing particle filter simulations with 8–8192 particles, we find that using greater numbers of particles consistently increases the likelihood of the whole AFM movie. By comparing the likelihoods for different ionic concentrations and time scale mappings, we find that the “true” concentration and time scale mapping can be inferred as the largest likelihood of the whole AFM movie but not that of each AFM image. The particle filter method provides a general approach for integrating HS-AFM data with MD simulations

Investigation into the role of LSH ATPase in chromatin remodelling

Chromatin remodelling is a crucial nuclear process affecting replication, transcription and repair. Global reduction of DNA methylation is observed in Immunodeficiency-Centromeric Instability-Facial Anomaly (ICF) syndrome. Several proteins were found to be mutated in patients diagnosed with ICF, among them are LSH and CDCA7. LSH is a chromatin remodeller bearing homology to the members of Sf2 remodelling family. A point mutation in its ATPase lobe was identified in ICF. CDCA7 is a zinc finger protein that was recently found to be crucial for nucleosome remodelling activity of LSH. Several point mutations in its zinc finger domain were described in ICF patients. In vitro and in vivo studies have shown that LSH-/- phenotype demonstrates reduction of global DNA methylation, implying that chromatin remodelling LSH functions may be required for the efficient methyltransferase activity, linking this finding to ICF phenotype. Here, the LSH-mononucleosome interaction was explored in vitro using bioinformatics, biochemical, biophysical and structural techniques. LSH purification was further optimised, achieving near 100% purity, which is a useful improvement for any potential structural studies. LSH has been found to interact with the mononucleosome in vitro and no DNA linker was required for this interaction, indicating that LSH binds to the nuclesomal core through its ATPase domain. Qualitatively estimated Kd for this interaction was in nanomolar region, which did not translate into complex detection during size exclusion chromatography. CDCA7 was expressed in insect cell system and semi-purified, however, high nucleic acid presence in the final protein sample precluded any potential studies of CDCA7 interaction with chromatin. Homology and ab initio modelling for LSH and CDCA7, respectively, indicated that LSH is likely to bind the superhelical location 2 (SHL2), however, the exact location of CDCA7 and its interaction with LSH will have to be elucidated in further experimental work

Single molecule investigation of transcription activator-like effector search dynamics

Recent advances in genetic engineering hold great potential to profoundly change the treatment of human disease. Precise manipulation of genetic material allows for the creation of new disease models and the rapid translation of new therapies into the clinic. Several classes of programmable nucleases allowing for precise and targeted genomic edits are central to the rising popularity, flexibility, and accessibility of gene engineering. Transcription activator-like effectors (TALEs) are one such class that form a powerful gene-editing platform when fused to nuclease domains, thereby yielding TALEN systems. Despite pervasive use of TALENs for editing crops, small animals, eukaryotic stem cells, and human T-cells, remarkably little is known about the molecular mechanisms used to locate and bind their DNA target sites. This work describes the application of single molecule fluorescence imaging to the study the TALE search process along specific and non-specific DNA templates. Our work provides a molecular-level picture of the dynamics of TALE-DNA interactions, and our results have revealed an apparently unique search mechanism for DNA binding proteins. We directly observe TALEs diffusing along non-specific DNA in one-dimension. Our results show that TALE diffusion occurs in a directionally unbiased and thermally driven manner along double surface tethered and extended DNA templates (Chapter 2). Interestingly, we observe significant intra-trajectory heterogeneity for diffusion of full-length TALE proteins. We further isolate and study the single molecule dynamics of TALE truncation mutants containing only the N-terminal region (NTR), and these results reveal the importance of the NTR for nucleating non-specific binding. We find that the TALE NTR alone is capable of short, rapid non-specific search. Furthermore, we study the diffusion of a series of TALEs with variable size central repeat domains (CRDs). Taken together with insights from NTR dynamics and the heterogeneity of full-length TALE diffusion, we propose a two-state search mechanism for TALEs that is comprised of rapid search and interspersed periods of local sequence checking along DNA templates. We further expand our characterization of TALE search by determining the impact of solution conditions, ionic strength, probe size, and the role of hydrodynamic flow on TALE dynamics (Chapter 3). Using this combination of single molecule experiments, we find that TALE diffusion does not fit the traditional definitions of binary classification of DNA-binding protein search, which have been characterized as protein hopping or sliding along DNA templates. Instead, our results suggest a mechanism wherein TALEs encircle DNA templates during search, but form only transient contacts with the DNA backbone. Furthermore, the non-specific search trajectory of TALEs is rotationally decoupled, in contrast to a broad class of other DNA binding proteins including DNA repair proteins and transcription factors. We further utilize a combination of bulk fluorescence anisotropy measurements and single molecule experiments to characterize the effects of divalent cations on TALE binding (Chapter 4). Our results show that TALE specificity is significantly enhanced in the presence of certain divalent cations, which can be attributed to a decrease in non-specific binding affinity. Finally, we generate long DNA templates with TALE target binding arrays at precise locations, and we directly visualize specific binding and localization of TALEs to their respective target sites following 1-D search. Taken together, our results have elucidated the fundamental search mechanism of TALE proteins along DNA templates