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

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.

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Supercoiling DNA locates mismatches

10.1101/130567 ◽

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.

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Analysis of the mechanism of DNA recombination using tangles

Quarterly Reviews of Biophysics ◽

10.1017/s0033583500003498 ◽

1995 ◽

Vol 28 (3) ◽

pp. 253-313 ◽

Cited By ~ 66

Author(s):

De Witt Sumners ◽

Claus Ernst ◽

Sylvia J. Spengler ◽

Nicholas R. Cozzarelli

Keyword(s):

Dna Recombination ◽

Dna Supercoiling ◽

Topological Properties ◽

Circular Dna ◽

Linear Dna ◽

Naturally Occurring

The DNA of all organisms has a complex and essential topology. The three topological properties of naturally occurring DNA are supercoiling, catenation, and knotting. Although these properties are denned rigorously only for closed circular DNA, even linear DNA in vivo can have topological properties because it is divided into topologically separate subdomains (Drlica 1987; Roberge & Gasser, 1992). The essentiality of topological properties is demonstrated by the lethal consequence of interfering with topoisomerases, the enzymes that regulate the level of DNA supercoiling and that unlink DNA during its replication (reviewed in Wang, 1991; Bjornsti, 1991; Drlica, 1992; Ullsperger et al. 1995).

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

10.1101/2019.12.20.885533 ◽

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.

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Supercoiling DNA optically

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1908826116 ◽

2019 ◽

Vol 116 (52) ◽

pp. 26534-26539

Author(s):

Graeme A. King ◽

Federica Burla ◽

Erwin J. G. Peterman ◽

Gijs J. L. Wuite

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Dna Supercoiling ◽

Biological Interactions ◽

Mitochondrial Transcription ◽

Supercoiled Dna ◽

Single Molecule Level ◽

Mitochondrial Transcription Factor ◽

Wide Range

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa836 ◽

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.

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Biomedical applications: Pitfalls in the practice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169626 ◽

1994 ◽

Vol 52 ◽

pp. 378-379

Author(s):

J. D. Shelburne ◽

Peter Ingram ◽

Victor L. Roggli ◽

Ann LeFurgey

Keyword(s):

Operating Room ◽

Liquid Nitrogen ◽

Lung Tissue ◽

Biomedical Applications ◽

Microprobe Analysis ◽

Tissue Sample ◽

Cellular Level ◽

Tissue Samples ◽

Asbestos Fibers

At present most medical microprobe analysis is conducted on insoluble particulates such as asbestos fibers in lung tissue. Cryotechniques are not necessary for this type of specimen. Insoluble particulates can be processed conventionally. Nevertheless, it is important to emphasize that conventional processing is unacceptable for specimens in which electrolyte distributions in tissues are sought. It is necessary to flash-freeze in order to preserve the integrity of electrolyte distributions at the subcellular and cellular level. Ideally, biopsies should be flash-frozen in the operating room rather than being frozen several minutes later in a histology laboratory. Electrolytes will move during such a long delay. While flammable cryogens such as propane obviously cannot be used in an operating room, liquid nitrogen-cooled slam-freezing devices or guns may be permitted, and are the best way to achieve an artifact-free, accurate tissue sample which truly reflects the in vivo state. Unfortunately, the importance of cryofixation is often not understood. Investigators bring tissue samples fixed in glutaraldehyde to a microprobe laboratory with a request for microprobe analysis for electrolytes.

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Exploiting Anti-Inflammation Effects of Flavonoids in Chronic Inflammatory Diseases

Current Pharmaceutical Design ◽

10.2174/1381612826666200408101550 ◽

2020 ◽

Vol 26 (22) ◽

pp. 2610-2619 ◽

Cited By ~ 2

Author(s):

Tarique Hussain ◽

Ghulam Murtaza ◽

Huansheng Yang ◽

Muhammad S. Kalhoro ◽

Dildar H. Kalhoro

Keyword(s):

Oxidative Stress ◽

Chronic Diseases ◽

Inflammatory Diseases ◽

Therapeutic Option ◽

Cellular Level ◽

Antiinflammatory Drugs ◽

Suitable Alternative ◽

Chronic Inflammatory Diseases

Background: Inflammation is a complex response of the host defense system to different internal and external stimuli. It is believed that persistent inflammation may lead to chronic inflammatory diseases such as, inflammatory bowel disease, neurological and cardiovascular diseases. Oxidative stress is the main factor responsible for the augmentation of inflammation via various molecular pathways. Therefore, alleviating oxidative stress is effective a therapeutic option against chronic inflammatory diseases. Methods: This review article extends the knowledge of the regulatory mechanisms of flavonoids targeting inflammatory pathways in chronic diseases, which would be the best approach for the development of suitable therapeutic agents against chronic diseases. Results: Since the inflammatory response is initiated by numerous signaling molecules like NF-κB, MAPK, and Arachidonic acid pathways, their encountering function can be evaluated with the activation of Nrf2 pathway, a promising approach to inhibit/prevent chronic inflammatory diseases by flavonoids. Over the last few decades, flavonoids drew much attention as a potent alternative therapeutic agent. Recent clinical evidence has shown significant impacts of flavonoids on chronic diseases in different in-vivo and in-vitro models. Conclusion: Flavonoid compounds can interact with chronic inflammatory diseases at the cellular level and modulate the response of protein pathways. A promising approach is needed to overlook suitable alternative compounds providing more therapeutic efficacy and exerting fewer side effects than commercially available antiinflammatory drugs.

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HG-9-91-01 Attenuates Murine Experimental Colitis by Promoting Interleukin-10 Production in Colonic Macrophages Through the SIK/CRTC3 Pathway

Inflammatory Bowel Diseases ◽

10.1093/ibd/izab072 ◽

2021 ◽

Author(s):

Yong Fu ◽

Gailing Ma ◽

Yuqian Zhang ◽

Wenli Wang ◽

Tongguo Shi ◽

...

Keyword(s):

Mononuclear Cells ◽

Experimental Colitis ◽

Underlying Mechanism ◽

Interleukin 10 ◽

Camp Response Element Binding ◽

Cellular Level ◽

Trinitrobenzene Sulfonic Acid ◽

Murine Colitis ◽

Anti Inflammatory

Abstract Background Interleukin-10 (IL-10) is a potent immunoregulatory cytokine that plays a pivotal role in maintaining mucosal immune homeostasis. As a novel synthetic inhibitor of salt-inducible kinases (SIKs), HG-9-91-01 can effectively enhance IL-10 secretion at the cellular level, but its in vivo immunoregulatory effects remain unclear. In this study, we investigated the effects and underlying mechanism of HG-9-91-01 in murine colitis models. Methods The anti-inflammatory effects of HG-9-91-01 were evaluated on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-, dextran sulfate sodium–induced colitis mice, and IL-10 knockout chronic colitis mice. The in vivo effector cell of HG-9-91-01 was identified by fluorescence-activated cell sorting and quantitative real-time polymerase chain reaction. The underlying mechanism of HG-9-91-01 was investigated via overexpressing SIKs in ANA-1 macrophages and TNBS colitis mice. Results Treatment with HG-9-91-01 showed favorable anticolitis effects in both TNBS- and DSS-treated mice through significantly promoting IL-10 expression in colonic macrophages but failed to protect against IL-10 KO murine colitis. Further study indicated that HG-9-91-01 markedly enhanced the nuclear level of cAMP response element-binding protein (CREB)-regulated transcription coactivator 3 (CRTC3), whereas treatment with lentiviruses encoding SIK protein markedly decreased the nuclear CRTC3 level in HG-9-91-01–treated ANA-1 macrophages. In addition, intracolonic administration with lentiviruses encoding SIK protein significantly decreased the nuclear CRTC3 level in the lamina propria mononuclear cells and ended the anti-inflammatory activities of HG-9-91-01. Conclusions We found that HG-9-91-01 promoted the IL-10 expression of colonic macrophages and exhibited its anticolitis activity through the SIK/CRTC3 axis, and thus it may represent a promising strategy for inflammatory bowel disease therapy.

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Cellular sources of TSPO expression in healthy and diseased brain

European Journal of Nuclear Medicine and Molecular Imaging ◽

10.1007/s00259-020-05166-2 ◽

2021 ◽

Author(s):

Erik Nutma ◽

Kelly Ceyzériat ◽

Sandra Amor ◽

Stergios Tsartsalis ◽

Philippe Millet ◽

...

Keyword(s):

Cell Activity ◽

Brain Regions ◽

Cellular Level ◽

Translocator Protein ◽

Disease Stage ◽

Animal Models Of Disease ◽

Positron Emission ◽

Neuropsychiatric Diseases ◽

Tspo Expression

AbstractThe 18 kDa translocator protein (TSPO) is a highly conserved protein located in the outer mitochondrial membrane. TSPO binding, as measured with positron emission tomography (PET), is considered an in vivo marker of neuroinflammation. Indeed, TSPO expression is altered in neurodegenerative, neuroinflammatory, and neuropsychiatric diseases. In PET studies, the TSPO signal is often viewed as a marker of microglial cell activity. However, there is little evidence in support of a microglia-specific TSPO expression. This review describes the cellular sources and functions of TSPO in animal models of disease and human studies, in health, and in central nervous system diseases. A discussion of methods of analysis and of quantification of TSPO is also presented. Overall, it appears that the alterations of TSPO binding, their cellular underpinnings, and the functional significance of such alterations depend on many factors, notably the pathology or the animal model under study, the disease stage, and the involved brain regions. Thus, further studies are needed to fully determine how changes in TSPO binding occur at the cellular level with the ultimate goal of revealing potential therapeutic pathways.

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