High‐throughput single‐molecule imaging system using nanofabricated trenches and fluorescent DNA‐binding proteins

2020 ◽  
Vol 117 (6) ◽  
pp. 1640-1648
Author(s):  
Yujin Kang ◽  
Na Young Cheon ◽  
Jongjin Cha ◽  
Ayoung Kim ◽  
Hyung‐il Kim ◽  
...  
Keyword(s):  
Dna Binding ◽  
High Throughput ◽  
Single Molecule ◽  
Binding Proteins ◽  
Imaging System ◽  
Dna Binding Proteins ◽  
Single Molecule Imaging

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 Sub-microsecond Single-Molecule FRET Studies of Single-Stranded DNA Conformation Fluctuations Mediated by Single-Stranded DNA Binding Proteins

2020 ◽  
Vol 118 (3) ◽  
pp. 188a
Author(s):  
Brett A. Israels ◽  
Anson Dang ◽  
Peter H. von Hippel ◽  
Andrew H. Marcus
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Dna Conformation ◽  
Single Stranded Dna ◽  
Single Molecule Fret

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.

'Calling Cards' method for high-throughput identification of targets of yeast DNA-binding proteins

2008 ◽  
Vol 3 (10) ◽  
pp. 1569-1577 ◽  
Author(s):  
Haoyi Wang ◽  
Michael E Heinz ◽  
Seth D Crosby ◽  
Mark Johnston ◽  
Robi D Mitra
Keyword(s):  
Dna Binding ◽  
High Throughput ◽  
Binding Proteins ◽  
Dna Binding Proteins

scholarly journals Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

2020 ◽  
Author(s):  
Mathew Stracy ◽  
Jakob Schweizer ◽  
David J Sherratt ◽  
Achillefs N Kapanidis ◽  
Stephan Uphoff ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Proteins ◽  
Search Time ◽  
Dna Binding Proteins ◽  
Protein Diffusion ◽  
Chromosomal Dna ◽  
Dna Interactions ◽  
Target Search ◽  
Molecular Weights

ABSTRACTDespite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Towards identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli. The mobility of these proteins during the target search was dictated by DNA interactions, rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid does not pose a physical barrier for protein diffusion, but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58-99%) of their search time bound to DNA and occupy as much as ∼30% of the chromosomal DNA at any time. Chromosome-crowding likely has important implications for the function of all DNA-binding proteins.

scholarly journals Single Molecule FRET Analysis of DNA Binding Proteins

2017 ◽  
pp. 217-239
Author(s):  
Kathy R. Chaurasiya ◽  
Remus T. Dame
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Single Molecule Fret

scholarly journals Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase

2018 ◽  
Author(s):  
Timothy D. Craggs ◽  
Marko Sustarsic ◽  
Anne Plochowietz ◽  
Majid Mosayebi ◽  
Hendrik Kaju ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Polymerase ◽  
Single Molecule ◽  
Solution Structure ◽  
Binding Proteins ◽  
Conformational Dynamics ◽  
Dna Binding Proteins ◽  
Binary Complex ◽  
Specific Recognition ◽  
Gapped Dna

AbstractDNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120°) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarsegrained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy (<ΔFRET> = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state (ΔG < 4 kT); such conformations were much less accessible to nicked (> 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.Significance StatementMost genetic processes, including DNA replication, repair and transcription, rely on DNA-binding proteins locating specific sites on DNA; some sites contain a specific sequence, whereas others present a specific structure. While sequence-specific recognition has a clear physical basis, structure-specific recognition mechanisms remain obscure. Here, we use single-molecule FRET and computer simulations to show that the conformational dynamics of an important repair intermediate (1nt-gapped DNA) act as central recognition signals for structure-specific binding by DNA polymerase I (Pol). Our conclusion is strongly supported by a novel solution structure of the Pol-DNA complex wherein the gapped-DNA is significantly bent. Our iterative approach combining precise single-molecule measurements with molecular modelling is general and can elucidate the structure and dynamics for many large biomachines.

scholarly journals Identification of multiple kinetic populations of DNA-binding proteins in live cells

2019 ◽  
Author(s):  
Han N. Ho ◽  
Daniel Zalami ◽  
Jürgen Köhler ◽  
Antoine M. van Oijen ◽  
Harshad Ghodke
Keyword(s):  
Dna Binding ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dynamic Range ◽  
Time Lapse ◽  
Dna Binding Proteins ◽  
Live Cells ◽  
Single Molecule Fluorescence ◽  
Time Lapse Imaging

ABSTRACTUnderstanding how multi-protein complexes function in cells requires detailed quantitative understanding of their association and dissociation kinetics. Analysis of the heterogeneity of binding lifetimes enables interrogation of the various intermediate states formed during the reaction. Single-molecule fluorescence imaging permits the measurement of reaction kinetics inside living organisms with minimal perturbation. However, poor photo-physical properties of fluorescent probes limit the dynamic range and accuracy of measurements of off rates in live cells. Time-lapse single-molecule fluorescence imaging can partially overcome the limits of photobleaching, however, limitations of this technique remain uncharacterized. Here, we present a structured analysis of which timescales are most accessible using the time-lapse imaging approach and explore uncertainties in determining kinetic sub-populations. We demonstrate the effect of shot noise on the precision of the measurements, as well as the resolution and dynamic range limits that are inherent to the method. Our work provides a convenient implementation to determine theoretical errors from measurements and to support interpretation of experimental data.STATEMENT OF SIGNIFICANCEMeasuring lifetimes of interactions between DNA-binding proteins and their substrates is important for understanding how they function in cells. In principle, time-lapse imaging of fluorescently-tagged proteins using single-molecule methods can be used to identify multiple sub-populations of DNA-binding proteins and determine binding lifetimes lasting for several tens of minutes. Despite this potential, currently available guidelines for the selection of binding models are unreliable, and the practical implementation of this approach is limited. Here, using experimental and simulated data we identify the minimum size of the dataset required to resolve multiple populations reliably and measure binding lifetimes with desired accuracy. This work serves to provide a guide to data collection, and measurement of DNA-binding lifetimes from single-molecule time-lapse imaging data.

DNA Garden: A Simple Method for Producing Arrays of Stretchable DNA for Single-Molecule Fluorescence Imaging of DNA-Binding Proteins

2017 ◽  
Vol 90 (1) ◽  
pp. 34-43 ◽  
Author(s):  
Chihiro Igarashi ◽  
Agato Murata ◽  
Yuji Itoh ◽  
Dwiky Rendra Graha Subekti ◽  
Satoshi Takahashi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Single Molecule Fluorescence ◽  
Simple Method
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