Role of Macromolecular Crowding on the Intracellular Diffusion of DNA Binding Proteins

Dey, Pinki; Bhattacherjee, Arnab

doi:10.1038/s41598-017-18933-3

Cited by 39 publications

(44 citation statements)

References 58 publications

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“…Besides molecular crowders, polyanionic DNA actively interacts with polypeptides and proteins, and this affects the T m . Polycations such as polylysine bind with a polyanionic DNA to form a complex (polyplex).…”

Section: Introductionmentioning

confidence: 99%

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

et al. 2018

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Nucleic acid hybridisation plays a key role in many biological processes, including transcription, translation, and regulation of gene expression. Several sophisticated applications rely on this fundamental interaction, including the polymerase chain reaction, sequencing, and gene therapy. To target a nucleic acid sequence specifically, synthetic oligonucleotides with a suitable affinity and specificity towards the target need to be designed. The affinity of potential probes or therapeutic agents to their target sequence is generally investigated by melting experiments, which break the hydrogen-bonding and stacking interactions that stabilise the double helix resulting in the formation of two single strands. In this paper, we report a comparative study of hybridisation for short fluorescent oligonucleotides labelled with cyanine and ATTO dyes, performed by the currently used UV melting assay and by a more sensitive fluorescence melting experiment. Using different oligonucleotide sequences in the concentration range of 5 nm to 2 μm, we observed a stabilising effect of the fluorophores on the duplexes, especially at low concentrations. We paid particular attention to the effect of polycations and to molecular crowding as major parameters that define the stability and the geometry of nucleic acid duplexes in biological samples. We also demonstrated how the fluorometry-based melting data could aid the design of a probe targeting a human BRAF gene fragment to reduce the off-target binding by a factor of seven.

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Section: Introductionmentioning

confidence: 99%

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

et al. 2018

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“…Further systematic investigation of diffusion in crowded media (22,37) supported the viewpoint and predicted that the role of bulk crowders is to prevent the dissociation of DBPs from the DNA surface and thereby promoting 1D diffusion of the searching protein along the DNA contour over 3D diffusion (38,39). In contrast, previous studies indicate that purely repulsive crowders play a role to decouple the searching protein from the crowding agents during the 1D search regime on a linear DNA stretch (40,41). The observation is along the line of Asakura-Oosawa model (AOM) (42) of crowder action, where the crowding agents, due to excluded volume interactions with the DNA molecule result in a preferentially depleted volume around the DNA interface.…”

Section: Introductionmentioning

confidence: 86%

“…The newly formed depletion zone is referred as composite depletion zone that features a narrower width because of attractive depletion force. Schematic description of the formation of composite depletion zone is depicted in Fig S2. To identify various search modes adopted by the diffusing protein, we follow the description similar to our previous works (40,41). Briefly, the protein is said to perform sliding if at least 70% of the recognition helix of Sap-1 is within the DNA major groove with an orientation angle of < 25 • , and the center of mass of the recognition helix lies within 8 Å to the closest DNA base pair in the absence of crowder molecules (see Fig S3).…”

Section: Estimation Of Depletion Layer Dimension and Characterizing Smentioning

confidence: 99%

“…The observation is along the line of Asakura-Oosawa model (AOM) (42) of crowder action, where the crowding agents, due to excluded volume interactions with the DNA molecule result in a preferentially depleted volume around the DNA interface. Since the region is devoid of crowder molecules, the nonspecific rotational and translational diffusion of the searching protein inside this region were found to be independent of the concentration of bulk crowders (40). The protein effectively experiences a constant microviscosity much lower than that of the bulk, which could promote faster diffusion of the protein.…”

Section: Introductionmentioning

confidence: 97%

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Structural Basis of Enhanced Facilitated Diffusion of DNA Binding Proteins in Crowded Cellular Milieu

Bhattarcharjee

Dey

2019

Preprint

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DNA binding proteins (DBPs) rapidly recognize and specifically associate with their target DNA sites inside cell nucleus that contains up to 400 g/L macromolecules, most of which are proteins. While the fast association between DBPs and DNA is explained by a facilitated diffusion mechanism, where DBPs adopt a weighted combination of 3D diffusion and 1D sliding and hopping modes of transportation, the role of cellular environment that contains many nonspecifically interacting proteins and other biomolecules is mostly overlooked. By performing large scale computational simulations with an appropriately tuned model of protein and DNA in the presence of nonspecifically interacting bulk and DNA bound crowders (genomic crowders), we demonstrate the structural basis of the enhanced facilitated diffusion of DBPs inside a crowded cellular milieu through novel 1D scanning mechanisms. In the presence of bulk crowders, we identify the protein to float along the DNA under the influence of protein-crowder nonspecific interactions. The search mode is distinctly different compared to usual 1D sliding and hopping dynamics where protein diffusion is regulated by the DNA electrostatics. In contrast, the presence of genomic crowders expedite the target search process by transporting the protein over DNA segments through the formation of a transient protein-crowder bridged complex. By analyzing the ruggedness of the associated potential energy landscape, we underpin the molecular origin of the kinetic advantages of these search modes and show that they successfully explain the experimentally observed acceleration of facilitated diffusion of DBPs by molecular crowding agents and crowder concentration dependent enzymatic activity of transcription factors. Our findings provide crucial insights into gene regulation kinetics inside the crowded cellular milieu.SIGNIFICANCE 10-40% of the intracellular volume is occupied by proteins, and other biomolecules, collectively known as macromolecular crowders. Their presence has been found to promote faster translocation of DNA binding proteins (DBPs) during the search of their target DNA sites for crucial cellular processes. Using molecular simulations, we probe the underlying structural basis and underscore the existence of novel DNA scanning mechanisms actuated by protein-crowder nonspecific interactions. We show that the observed search modes are kinetically beneficial and can successfully explain the acceleration of facilitated diffusion of DBPs by molecular crowding agents and crowderconcentration dependent enzymatic activity of transcription factors.Our study sheds new light on the long-standing facilitated diffusion problem of DBPs in the crowded cellular environment for regulating gene expression.

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“…We adopted a coarse-grained representation of protein, where each amino acid is represented by a single bead placed at the respective Cα position (34). The energetics of the protein is described by a structure-based potential (35) (for details, see the Supporting text), which represents the funnel-like energy landscape for protein folding (35) and has been used extensively in studying protein-protein (36) and protein-nucleic acid interactions (37)(38)(39)(40)(41)(42)(43). The resolution of the ssDNA molecule is three beads per nucleotide (phosphate, sugar and nitrogenous base), placed at their respective geometric centers.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of Dynamic Binding of Replication Protein A to ssDNA

Bhattarcharjee

Mondal

2020

Preprint

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Replication protein A (RPA) serves as hub protein inside eukaryotic cells, where it coordinates crucial DNA metabolic processes and activates the DNA-damage response system. A characteristic feature of its action is to associate with ssDNA intermediates before handing over them to downstream proteins. The length of ssDNA intermediates differs for different pathways. This means RPA must have mechanisms for selective processing of ssDNA intermediates based on their length, the knowledge of which is fundamental to elucidate when and how DNA repair and replication processes are symphonized. By employing extensive molecular simulations, we investigated the mechanism of binding of RPA to ssDNA of different lengths. We show that the binding involves dynamic equilibrium with a stable intermediate, the population of which increases with the length of ssDNA. The vital underlying factors are decoded through collective variable principal component analysis. It suggests a differently orchestrated set of interactions that define the action of RPA based on the sizes of ssDNA intermediates. We further estimated the association kinetics and probed the diffusion mechanism of RPA to ssDNA. RPA diffuses on short ssDNA through progressive 'bulge' formation. With long ssDNA, we observed a conformational change in ssDNA coupled with its binding to RPA in a cooperative fashion. Our analysis explains how the 'short-lived,' long ssDNA intermediates are processed quickly in vivo. The study thus reveals the molecular basis of several recent experimental observations related to RPA binding to ssDNA and provides novel insights into the RPA functioning in DNA repair and replication. Significance StatementDespite ssDNA be the common intermediate to all pathways involving RPA, how does the latter function differently in the DNA processing events such as DNA repair, replication, and recombination just based on the length of ssDNA intermediates remains unknown. The major hindrance is the difficulty in capturing the transient interactions between the molecules. Even attempts to crystallize RPA complexes with 32nt and 62nt ssDNA have yielded a resolved structure of only 25nt ssDNA wrapped with RPA. Here, we used a state-of-the-art coarse-grained protein-ssDNA model to unravel the detailed mechanism of binding of RPA to ssDNA. Our study illustrates the molecular origin of variations in RPA action during various DNA processing events depending on the length of ssDNA intermediates.

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Role of Macromolecular Crowding on the Intracellular Diffusion of DNA Binding Proteins

Cited by 39 publications

References 58 publications

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

Structural Basis of Enhanced Facilitated Diffusion of DNA Binding Proteins in Crowded Cellular Milieu

Mechanism of Dynamic Binding of Replication Protein A to ssDNA

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