scholarly journals Facilitated Dissociation of Nucleoid Associated Proteins from DNA in the Bacterial Confinement

2021 ◽  
Author(s):  
Zafer Koşar ◽  
A. Göktuĝ Attar ◽  
Aykut Erbaş
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Experimental Studies ◽  
Dual Purpose ◽  
E Coli ◽  
Protein Dna Interactions ◽  
Factor For Inversion Stimulation ◽  
Associated Proteins ◽  
Order Of Magnitude ◽  
Single Molecule Studies

Transcription machinery ultimately depends on the temporal formation of protein-DNA complexes. Recent experimental studies demonstrate that residence time (i.e., inverse off-rate) of a transcription factor protein can be a contributor to the functional diversity of the protein. In the meantime, single-molecule experiments showed that the off-rates of a wide array of DNA-binding proteins accelerate as the bulk concentration of the protein increases via a concentration-dependent mechanism (i.e., facilitated dissociation, FD). In this study, inspired by the previous single-molecule studies on the factor for inversion stimulation (Fis) protein of E. coli, which is a dual-purpose protein with a diverse functionality, we model the unbinding of Fis from specific bindings sites along a high-molecular-weight circular DNA in a cylindrical structure mimicking the cellular confinement of chromosome. Our simulations show that FD of Fis can well occur in confinement at physiological concentrations. Particularly, when nutrient-rich conditions are emulated with Fis concentrations around micromolar levels, the off-rates increase one order of magnitude compared to the lower Fis levels. However, Fis significantly changes the chromosome structure at higher concentrations by forming dense protein clusters bridging specific sites and juxtaposing remote DNA segments. As a result, at the physiologically observed maximum levels of Fis, the off-rates significantly slow down. Overall, our results indicate that cellular-concentration levels of a structural DNA-binding protein is intermingled with the genome architecture and DNA residence times, thereby providing a basis for understanding the complex effects of dynamic protein-DNA interactions on gene regulation.

scholarly journals Single Molecule Studies of DNA-Binding Proteins: Development of New Covalent DNA Anchoring Techniques for the Study of Rupture Forces of Replication Blocks

2014 ◽  
Vol 106 (2) ◽  
pp. 394a
Author(s):  
Richard Janissen ◽  
Bojk A. Berghuis ◽  
Orkide Ordu ◽  
Max M. Wink ◽  
David Dulin ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Single Molecule Studies

scholarly journals The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA

2020 ◽  
Vol 48 (19) ◽  
pp. 10820-10831
Author(s):  
Kiyoto Kamagata ◽  
Kana Ouchi ◽  
Cheng Tan ◽  
Eriko Mano ◽  
Sridhar Mandali ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Modes ◽  
Dna Structures ◽  
Bacterial Proteins ◽  
Associated Proteins ◽  
Diffusion Mechanisms ◽  
Dynamics Simulations

Abstract DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.

scholarly journals Switch-like control of helicase processivity by single-stranded DNA binding protein

2020 ◽  
Author(s):  
Barbara Stekas ◽  
Masayoshi Honda ◽  
Maria Spies ◽  
Yann R. Chemla
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Optical Trap ◽  
Dna Binding Protein ◽  
Single Stranded Dna ◽  
Associated Proteins ◽  
Underlying Mechanisms ◽  
Xpd Helicase

Helicases utilize the energy of NTP hydrolysis to translocate along single-stranded nucleic acids (NA) and unwind the duplex. In the cell, helicases function in the context of other NA-associated proteins which regulate helicase function. For example, single-stranded DNA binding proteins are known to enhance helicase activity, although the underlying mechanisms remain largely unknown. F. acidarmanus XPD helicase serves as a model for understanding the molecular mechanisms of Superfamily 2B helicases, and previous work has shown that its activity is enhanced by the cognate single-stranded DNA binding protein RPA2. Here, single-molecule optical trap measurements of the unwinding activity of a single XPD helicase in the presence of RPA2 reveal a mechanism in which XPD interconverts between two states with different processivities and transient RPA2 interactions stabilize the more processive state, activating a latent “processivity switch” in XPD. These findings provide new insights on mechanisms of helicase regulation by accessory proteins.

scholarly journals Impact of Small Molecules on Intermolecular G-Quadruplex Formation

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1570 ◽  
Author(s):  
Prabesh Gyawali ◽  
Keshav GC ◽  
Yue Ma ◽  
Sanjaya Abeysirigunawardena ◽  
Kazuo Nagasawa ◽  
...  
Keyword(s):  
Small Molecules ◽  
Single Molecule ◽  
The Other ◽  
Single Molecule Level ◽  
G Quadruplex ◽  
Dna Strands ◽  
Order Of Magnitude ◽  
Single Molecule Studies ◽  
Quadruplex Formation ◽  
The Impact

We performed single molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC3) on intermolecular G-quadruplex (i-GQ) formation between two guanine-rich DNA strands that had 3-GGG repeats in one strand and 1-GGG repeat in the other (3+1 GGG), or 2-GGG repeats in each strand (2+2 GGG). Such structures are not only physiologically significant but have recently found use in various biotechnology applications, ranging from DNA-based wires to chemical sensors. Understanding the extent of stability imparted by small molecules on i-GQ structures, has implications for these applications. The small molecules resulted in different levels of enhancement in i-GQ formation, depending on the small molecule and arrangement of GGG repeats. The largest enhancement we observed was in the 3+1 GGG arrangement, where i-GQ formation increased by an order of magnitude, in the presence of L2H2-6OTD. On the other hand, the enhancement was limited to three-fold with Pyridostatin (PDS) or less for the other small molecules in the 2+2 GGG repeat case. By demonstrating detection of i-GQ formation at the single molecule level, our studies illustrate the feasibility to develop more sensitive sensors that could operate with limited quantities of materials.

scholarly journals Single-Molecule Studies of the ssDNA Binding Activity of E. Coli MutL

2010 ◽  
Vol 98 (3) ◽  
pp. 64a-65a
Author(s):  
Jonghyun Park ◽  
Yong-Moon Jeon ◽  
Daekil In ◽  
Seong-Dal Heo ◽  
Changill Ban ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Activity ◽  
Ssdna Binding ◽  
E Coli ◽  
Single Molecule Studies

scholarly journals An Ultra-Stable and Dense Single-Molecule Click Platform for Sensing Protein-DNA Interactions

2020 ◽  
Author(s):  
Emiel W. A. Visser ◽  
Jovana Miladinovic ◽  
Joshua N. Milstein
Keyword(s):  
Single Molecule ◽  
Elevated Temperatures ◽  
Regulatory Protein ◽  
Bacterial Protein ◽  
Dna Interactions ◽  
Temperature Dependent ◽  
E Coli ◽  
Tethered Particle Motion ◽  
Protein Dna Interactions ◽  
Ultrahigh Density

AbstractWe demonstrate an ultra-stable, highly dense single-molecule assay ideal for observing protein-DNA interactions. Stable click Tethered Particle Motion (scTPM) leverages next generation click-chemistry to achieve an ultrahigh density of surface tethered reporter particles, has a high antifouling resistance, is stable at elevated temperatures to at least 45 °C, and is compatible with Mg2+, an important ionic component of many regulatory protein-DNA interactions. Prepared samples remain stable, with little degradation, for > 6 months in physiological buffers. These improvements enabled us to study previously inaccessible sequence and temperature dependent effects on DNA binding by the bacterial protein H-NS, a global transcriptional regulator found in E. Coli. This greatly improved assay can directly be translated to accelerate existing tethered particle based, single-molecule biosensing applications.

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