Searching target sites on DNA by proteins: Role of DNA dynamics under confinement

Mondal, Anupam; Bhattacherjee, Arnab

doi:10.1093/nar/gkv931

Cited by 46 publications

(57 citation statements)

References 84 publications

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“…Although 1D diffusion of Cas9 does not require external energy, we captured a surprisingly biased 1D diffusion of Cas9 on dsDNA under physiological buffer condition. Low-salt concentration, which strengthens interactions between DNA binding proteins and dsDNA (Berg et al, 1981;Bonnet et al, 2008;Lohman, 1986;Mondal and Bhattacherjee, 2015;Tempestini et al, 2018), causes Cas9 to display unbiased 1D diffusion. In addition, Cas9-VQR with altered PAM preference exhibits biased diffusion while Cas9-EQR unbiased diffuses on dsDNA under physiological salt condition (Fig.…”

Section: Discussionmentioning

confidence: 99%

Streptococcus pyogenes Cas9 displays biased one-dimensional diffusion on dsDNA to search for a target

Yang

Zhang

Sun

et al. 2019

Preprint

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The RNA-guided Streptococcus pyogenes Cas9 (spCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how the Cas9 locates its target among huge amounts of dsDNAs remains controversial. Here, combining biochemical and single-molecule assays, we revealed that Cas9 uses both three-dimensional and one-dimensional diffusion to find its target. We further observed a surprising biased one-dimensional diffusion of Cas9 from 3' to 5' end of the non-target strand under physiological salt condition, whereas low ionic concentration or mutations on PAM recognition residues induce unbiased one-dimensional diffusion of Cas9 along dsDNA. We quantified the diffusion length of 27 bp, which accelerates the target search efficiency of Cas9 by ~ 10 folds. Our results reveal a unique searching mechanism of Cas9 at physiological salt conditions, and provide important guidance for both in-vitro and in-vivo applications of Cas9.

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Section: Discussionmentioning

confidence: 99%

Streptococcus pyogenes Cas9 displays biased one-dimensional diffusion on dsDNA to search for a target

Yang

Zhang

Sun

et al. 2019

Preprint

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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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“…The role of cell confinement on the search of the DNA site was explored by Mondal and Bhattacherjee (2015) using a coarse-grained model of a 200-bp DNA, a coarse-grained DBP, and a sphero-cylindrical potential mimicking a prokaryotic cell. The authors found that both DNA flexibility and confinement increase the probability of binding between the DBP and the DNA.…”

Section: Computational Models Of the Genetic Materialsmentioning

confidence: 99%

Molecular simulations of cellular processes

Trovato

Fumagalli

2017

Biophys Rev

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It is, nowadays, possible to simulate biological processes in conditions that mimic the different cellular compartments. Several groups have performed these calculations using molecular models that vary in performance and accuracy. In many cases, the atomistic degrees of freedom have been eliminated, sacrificing both structural complexity and chemical specificity to be able to explore slow processes. In this review, we will discuss the insights gained from computer simulations on macromolecule diffusion, nuclear body formation, and processes involving the genetic material inside cell-mimicking spaces. We will also discuss the challenges to generate new models suitable for the simulations of biological processes on a cell scale and for cellcycle-long times, including non-equilibrium events such as the co-translational folding, misfolding, and aggregation of proteins. A prominent role will be played by the wise choice of the structural simplifications and, simultaneously, of a relatively complex energetic description. These challenging tasks will rely on the integration of experimental and computational methods, achieved through the application of efficient algorithms.

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Searching target sites on DNA by proteins: Role of DNA dynamics under confinement

Cited by 46 publications

References 84 publications

Streptococcus pyogenes Cas9 displays biased one-dimensional diffusion on dsDNA to search for a target

Streptococcus pyogenes Cas9 displays biased one-dimensional diffusion on dsDNA to search for a target

Mechanism of Dynamic Binding of Replication Protein A to ssDNA

Molecular simulations of cellular processes

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