scholarly journals Coarse-Grained Modelling of DNA Plectoneme Pinning in the Presence of Base-Pair Mismatches

2019 ◽  
Author(s):  
Parth Rakesh Desai ◽  
Sumitabha Brahmachari ◽  
John F. Marko ◽  
Siddhartha Das ◽  
Keir C. Neuman
Keyword(s):  
Salt Concentration ◽  
Mechanical Response ◽  
Dna Supercoiling ◽  
Coarse Grained ◽  
Base Pairs ◽  
Mismatched Dna ◽  
Double Stranded Dna ◽  
Theoretical Predictions ◽  
Statistical Mechanical

ABSTRACTDamaged or mismatched DNA bases result in the formation of physical defects in double-stranded DNA. In vivo, defects in DNA must be rapidly and efficiently repaired to maintain cellular function and integrity. Defects can also alter the mechanical response of DNA to bending and twisting constraints, both of which are important in defining the mechanics of DNA supercoiling. Here, we use coarse-grained molecular dynamics (MD) simulation and supporting statistical-mechanical theory to study the effect of mismatched base pairs on DNA supercoiling. Our simulations show that plectoneme pinning at the mismatch site is deterministic under conditions of relatively high force (> 2 pN) and high salt concentration (> 0.5 M NaCl). Under physiologically relevant conditions of lower force (0.3 pN) and lower salt concentration (0.2 M NaCl), we find that plectoneme pinning becomes probabilistic and the pinning probability increases with the mismatch size. These findings are in line with experimental observations. The simulation framework, validated with experimental results and supported by the theoretical predictions, provides a way to study the effect of defects on DNA supercoiling and the dynamics of supercoiling in molecular detail.

scholarly journals Coarse-grained modelling of DNA plectoneme pinning in the presence of base-pair mismatches

2020 ◽  
Vol 48 (19) ◽  
pp. 10713-10725
Author(s):  
Parth Rakesh Desai ◽  
Sumitabha Brahmachari ◽  
John F Marko ◽  
Siddhartha Das ◽  
Keir C Neuman
Keyword(s):  
Salt Concentration ◽  
Mechanical Response ◽  
Dna Supercoiling ◽  
Coarse Grained ◽  
Base Pairs ◽  
Mismatched Dna ◽  
Double Stranded Dna ◽  
Theoretical Predictions ◽  
Statistical Mechanical

Abstract Damaged or mismatched DNA bases result in the formation of physical defects in double-stranded DNA. In vivo, defects in DNA must be rapidly and efficiently repaired to maintain cellular function and integrity. Defects can also alter the mechanical response of DNA to bending and twisting constraints, both of which are important in defining the mechanics of DNA supercoiling. Here, we use coarse-grained molecular dynamics (MD) simulation and supporting statistical-mechanical theory to study the effect of mismatched base pairs on DNA supercoiling. Our simulations show that plectoneme pinning at the mismatch site is deterministic under conditions of relatively high force (>2 pN) and high salt concentration (>0.5 M NaCl). Under physiologically relevant conditions of lower force (0.3 pN) and lower salt concentration (0.2 M NaCl), we find that plectoneme pinning becomes probabilistic and the pinning probability increases with the mismatch size. These findings are in line with experimental observations. The simulation framework, validated with experimental results and supported by the theoretical predictions, provides a way to study the effect of defects on DNA supercoiling and the dynamics of supercoiling in molecular detail.

scholarly journals Supercoiling DNA locates mismatches

2017 ◽  
Author(s):  
Andrew Dittmore ◽  
Sumitabha Brahmachari ◽  
Yasuhara Takagi ◽  
John F. Marko ◽  
Keir C. Neuman
Keyword(s):  
Dna Sequence ◽  
Base Pair ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Relative Probability ◽  
Base Pairs ◽  
Damage Sensing ◽  
Mismatched Base Pair ◽  
Monovalent Salt

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.5 pN force and show that, under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

scholarly journals Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage

2020 ◽  
Vol 295 (47) ◽  
pp. 15933-15947
Author(s):  
Yu Xu ◽  
Akanksha Manghrani ◽  
Bei Liu ◽  
Honglue Shi ◽  
Uyen Pham ◽  
...  
Keyword(s):  
Dynamic Equilibrium ◽  
Alkylating Agents ◽  
Dimethyl Sulfate ◽  
Dot Blot ◽  
Base Pairs ◽  
Double Stranded Dna ◽  
Genome Wide ◽  
Damage Susceptibility

As the Watson–Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson–Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson–Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson–Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson–Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson–Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson–Crick A-T base pairs in dsDNA only conferred ∼130-fold protection against adenine-N1 methylation, and this protection was reduced to ∼40-fold for A(syn)-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson–Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.

In vitro excision of adeno-associated virus DNA from recombinant plasmids: isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

1988 ◽  
Vol 8 (6) ◽  
pp. 2513-2522
Author(s):  
J Gottlieb ◽  
N Muzyczka
Keyword(s):  
Dna Sequences ◽  
Base Pairs ◽  
Adeno Associated Virus ◽  
Recombinant Plasmids ◽  
Culture Cells ◽  
Double Stranded Dna ◽  
Enzyme Fraction ◽  
Nuclear Extracts

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, we isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G.C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G.C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

scholarly journals Interaction of the Escherichia coli HU Protein with Various Topological Forms of DNA

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1724
Author(s):  
Li Huang ◽  
Zhenfeng Zhang ◽  
Roger McMacken
Keyword(s):  
Dna Supercoiling ◽  
Cellular Level ◽  
Base Pairs ◽  
Supercoiled Dna ◽  
Linear Dna ◽  
Hu Protein ◽  
Binding Preference ◽  
Mass Ratios ◽  
Superhelical Density

E. coli histone-like protein HU has been shown to interact with different topological forms of DNA. Using radiolabeled HU, we examine the effects of DNA supercoiling on HU–DNA interactions. We show that HU binds preferentially to negatively supercoiled DNA and that the affinity of HU for DNA increases with increases in the negative superhelical density of DNA. Binding of HU to DNA is most sensitively influenced by DNA supercoiling within a narrow but physiologically relevant range of superhelicity (σ = −0.06–0). Under stoichiometric binding conditions, the affinity of HU for negatively supercoiled DNA (σ = −0.06) is more than 10 times higher than that for relaxed DNA at physiologically relevant HU/DNA mass ratios (e.g., 1:10). This binding preference, however, becomes negligible at HU/DNA mass ratios higher than 1:2. At saturation, HU binds both negatively supercoiled and relaxed DNA with similar stoichiometries, i.e., 5–6 base pairs per HU dimer. In our chemical crosslinking studies, we demonstrate that HU molecules bound to negatively supercoiled DNA are more readily crosslinked than those bound to linear DNA. At in vivo HU/DNA ratios, HU appears to exist predominantly in a tetrameric form on negatively supercoiled DNA and in a dimeric form on linear DNA. Using a DNA ligase-mediated nick closure assay, we show that approximately 20 HU dimers are required to constrain one negative supercoil on relaxed DNA. Although fewer HU dimers may be needed to constrain one negative supercoil on negatively supercoiled DNA, our results and estimates of the cellular level of HU argue against a major role for HU in constraining supercoils in vivo. We discuss our data within the context of the dynamic distribution of the HU protein in cells, where temporal and local changes of DNA supercoiling are known to take place.

A STRUCTURE-MOTIVATED MODEL OF THE PASSIVE MECHANICAL RESPONSE OF THE PRIMARY PORCINE RENAL ARTERY

2014 ◽  
Vol 14 (03) ◽  
pp. 1450033 ◽  
Author(s):  
BORAN ZHOU ◽  
LAUREN WOLF ◽  
ALEXANDER RACHEV ◽  
TAREK SHAZLY
Keyword(s):  
Renal Artery ◽  
Arterial Wall ◽  
Mechanical Response ◽  
Physiological Function ◽  
Functional Dependence ◽  
Stretch Ratio ◽  
Comprehensive Description ◽  
Axial Stretch ◽  
Theoretical Predictions

The primary renal arteries transport up to one fourth of cardiac output to the kidneys for blood plasma ultrafiltration, with a functional dependence on the vessel geometry, composition and mechanical properties. Despite the critical physiological function of the renal artery, the few biomechanical studies that have focused on this vessel are either uniaxial or only partially describe its bi-axial mechanical behavior. In this study, we quantify the passive mechanical response of the primary porcine renal artery through bi-axial mechanical testing that probes the pressure-deformed diameter and pressure-axial force relationships at various longitudinal extensions, including the in-vivo axial stretch ratio. Mechanical data are used to parameterize and validate a structure-motivated constitutive model of the arterial wall. Together, experimental data and theoretical predictions of the stress distribution within the arterial wall provide a comprehensive description of the passive mechanical response of the porcine renal artery.

scholarly journals In vitro excision of adeno-associated virus DNA from recombinant plasmids: isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences.

1988 ◽  
Vol 8 (6) ◽  
pp. 2513-2522 ◽  
Author(s):  
J Gottlieb ◽  
N Muzyczka
Keyword(s):  
Dna Sequences ◽  
Base Pairs ◽  
Adeno Associated Virus ◽  
Recombinant Plasmids ◽  
Culture Cells ◽  
Double Stranded Dna ◽  
Enzyme Fraction ◽  
Nuclear Extracts

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, we isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G.C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G.C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

scholarly journals From quantum to subcellular scales: multi-scale simulation approaches and the SIRAH force field

2019 ◽  
Vol 9 (3) ◽  
pp. 20180085 ◽  
Author(s):  
Matías R. Machado ◽  
Ari Zeida ◽  
Leonardo Darré ◽  
Sergio Pantano
Keyword(s):  
Force Field ◽  
Computational Methods ◽  
Coarse Grained ◽  
Cellular Biology ◽  
Base Pairs ◽  
Fine Grained ◽  
Multi Scale ◽  
Double Stranded Dna ◽  
Structural Methods ◽  
Simulation Scheme

Modern molecular and cellular biology profits from astonishing resolution structural methods, currently even reaching the whole cell level. This is encompassed by the development of computational methods providing a deep view into the structure and dynamics of molecular processes happening at very different scales in time and space. Linking such scales is of paramount importance when aiming at far-reaching biological questions. Computational methods at the interface between classical and coarse-grained resolutions are gaining momentum with several research groups dedicating important efforts to their development and tuning. An overview of such methods is addressed herein, with special emphasis on the SIRAH force field for coarse-grained and multi-scale simulations. Moreover, we provide proof of concept calculations on the implementation of a multi-scale simulation scheme including quantum calculations on a classical fine-grained/coarse-grained representation of double-stranded DNA. This opens the possibility to include the effect of large conformational fluctuations in chromatin segments on, for instance, the reactivity of particular base pairs within the same simulation framework.

scholarly journals A Quantitative Investigation on the Peripheral Nerve Response within the Small Strain Range

2019 ◽  
Vol 9 (6) ◽  
pp. 1115 ◽  
Author(s):  
Elisabetta Giannessi ◽  
Maria Stornelli ◽  
Alessandra Coli ◽  
Pier Sergi
Keyword(s):  
Peripheral Nerves ◽  
Mechanical Response ◽  
Strain Range ◽  
Small Strain ◽  
The Body ◽  
Nerve Response ◽  
Theoretical Predictions ◽  
The Central Nervous System

Peripheral nerves are very complex biological structures crucial to linking the central nervous system to the periphery of the body. However, their real behaviour is partially unknown because of the intrinsic difficulty of studying these structures in vivo. As a consequence, theoretical and computational tools together with in vitro experiments are widely used to approximate the mechanical response of the peripheral nervous tissue to different kind of solicitations. More specifically, particular conditions narrow the mechanical response of peripheral nerves within the small strain regime. Therefore, in this work, the mechanical response of nerves was investigated through the study of the relationships among strain, stress and displacements within the small strain range. Theoretical predictions were quantitatively compared to experimental evidences, while the displacement field was studied for different values of the tissue compressibility. This framework provided a straightforward computational assessment of the nerve response, which was needed to design suitable connections to biomaterials or neural interfaces within the small strain range.

scholarly journals Nonequilibrium dynamics and action at a distance in transcriptionally driven DNA supercoiling

2021 ◽  
Vol 118 (10) ◽  
pp. e1905215118
Author(s):  
Yair A. G. Fosado ◽  
Davide Michieletto ◽  
Chris A. Brackley ◽  
Davide Marenduzzo
Keyword(s):  
Brownian Dynamics ◽  
Dna Supercoiling ◽  
Three Dimensions ◽  
Coarse Grained ◽  
Nonequilibrium Dynamics ◽  
Relaxation Dynamics ◽  
Double Stranded Dna ◽  
Action At A Distance ◽  
Dynamics Simulations ◽  
Nucleotide Resolution

We study the effect of transcription on the kinetics of DNA supercoiling in three dimensions by means of Brownian dynamics simulations of a single-nucleotide–resolution coarse-grained model for double-stranded DNA. By explicitly accounting for the action of a transcribing RNA polymerase (RNAP), we characterize the geometry and nonequilibrium dynamics of the ensuing twin supercoiling domains. Contrary to the typical textbook picture, we find that the generation of twist by RNAP results in the formation of plectonemes (writhed DNA) some distance away. We further demonstrate that this translates into an “action at a distance” on DNA-binding proteins; for instance, positive supercoils downstream of an elongating RNAP destabilize nucleosomes long before the transcriptional machinery reaches the histone octamer. We also analyze the relaxation dynamics of supercoiled double-stranded DNA, and characterize the widely different timescales of twist diffusion, which is a simple and fast process, and writhe relaxation, which is much slower and entails multiple steps.

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