scholarly journals Force determination in lateral magnetic tweezers combined with TIRF microscopy

Nanoscale ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 4579-4590 ◽  
Author(s):  
J. Madariaga-Marcos ◽  
S. Hormeño ◽  
C. L. Pastrana ◽  
G. L. M. Fisher ◽  
M. S. Dillingham ◽  
...  
Keyword(s):  
Dna Binding ◽  
Magnetic Tweezers ◽  
Constant Force ◽  
Dna Molecules ◽  
Tirf Microscopy

We have designed and calibrated a magnetic tweezers module to laterally stretch DNA molecules at a constant force, which can be incorporated into conventional magnetic tweezers. We demonstrate the combination of lateral magnetic tweezers with TIRF microscopy by characterizing DNA binding by ParB.

scholarly journals ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

2018 ◽  
Author(s):  
Julene Madariaga-Marcos ◽  
Cesar L. Pastrana ◽  
Gemma L. M. Fisher ◽  
Mark S. Dillingham ◽  
Fernando Moreno-Herrero
Keyword(s):  
Dna Binding ◽  
Protein Binding ◽  
Network Formation ◽  
Critical Role ◽  
Magnetic Tweezers ◽  
Dna Condensation ◽  
Full Length ◽  
Tirf Microscopy ◽  
Fluorescence Measurements

AbstractBacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. In our previous work (Fisher et al., 2017), we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks. However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualize ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerization with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks.Significance StatementUsing combined Magnetic Tweezers and TIRF microscopy we show that the CTD of ParB blocks ParB network formation by heterodimerization with the full-length protein, which remains bound to the DNA.

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

The Analyst ◽  
2019 ◽  
Vol 144 (3) ◽  
pp. 921-927 ◽  
Author(s):  
Jihyun Park ◽  
Seonghyun Lee ◽  
Nabin Won ◽  
Eunji Shin ◽  
Soo-Hyun Kim ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Physical Map ◽  
Dna Molecules ◽  
Single Dna Molecules

Two-color DNA physical map for efficient identification of single DNA molecules.

scholarly journals Single molecule force spectroscopy studies of DNA denaturation by T4 gene 32 protein

2004 ◽  
Vol 18 (2) ◽  
pp. 203-211 ◽  
Author(s):  
Mark C. Williams ◽  
Kiran Pant ◽  
Ioulia Rouzina ◽  
Richard L. Karpel
Keyword(s):  
Dna Binding ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Protein Interactions ◽  
Force Spectroscopy ◽  
Melting Transition ◽  
Dna Molecules ◽  
Force Extension ◽  
Single Molecule Force Spectroscopy ◽  
Gene 32

Single molecule force spectroscopy is an emerging technique that can be used to measure the biophysical properties of single macromolecules such as nucleic acids and proteins. In particular, single DNA molecule stretching experiments are used to measure the elastic properties of these molecules and to induce structural transitions. We have demonstrated that double‒stranded DNA molecules undergo a force‒induced melting transition at high forces. Force–extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review the evidence of DNA melting in these experiments and discuss the example of DNA force‒induced melting in the presence of the single‒stranded DNA binding protein T4 gene 32. We show that this force spectroscopy technique is a useful probe of DNA–protein interactions, which allows us to obtain binding rates and binding free energies for these interactions.

scholarly journals DNA binding fluorescent proteins for the direct visualization of large DNA molecules

2015 ◽  
Vol 44 (1) ◽  
pp. e6-e6 ◽  
Author(s):  
Seonghyun Lee ◽  
Yeeun Oh ◽  
Jungyoon Lee ◽  
Sojeong Choe ◽  
Sangyong Lim ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fluorescent Proteins ◽  
Direct Visualization ◽  
Dna Molecules

scholarly journals Studies of the Compaction Mechanisms of DNA-Binding Proteins using Horizontal Magnetic Tweezers

2015 ◽  
Vol 108 (2) ◽  
pp. 73a
Author(s):  
Roberto Fabian ◽  
Christopher Tyson ◽  
Abhijit Sarkar
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Magnetic Tweezers ◽  
Dna Binding Proteins

scholarly journals Dynamics of DNA nicking and unwinding by the RepC–PcrA complex

2020 ◽  
Vol 48 (4) ◽  
pp. 2013-2025 ◽  
Author(s):  
Carolina Carrasco ◽  
Cesar L Pastrana ◽  
Clara Aicart-Ramos ◽  
Sanford H Leuba ◽  
Saleem A Khan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Antibiotic Resistance Genes ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Common Mechanism ◽  
Rolling Circle Replication ◽  
Rolling Circle ◽  
Gram Positive Bacteria ◽  
Dna Nicking ◽  
Plasmid Pt181

Abstract The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s−1, while a typical velocity of 50 bp s−1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.

Generation and release of DNA-binding vesicles by Haemophilus influenzae during induction and loss of competence

1982 ◽  
Vol 152 (2) ◽  
pp. 855-864
Author(s):  
R A Deich ◽  
L C Hoyer
Keyword(s):  
Dna Binding ◽  
Haemophilus Influenzae ◽  
Divalent Cations ◽  
Outer Membrane Proteins ◽  
Normal Growth ◽  
Growth Conditions ◽  
Binding Activity ◽  
Stable Form ◽  
Bacterial Cells ◽  
Dna Molecules

Genetic transformation of bacterial cells required the induction of a state of competence to bind and absorb free DNA molecules. Induction of competence in Haemophilus influenzae was accompanied by the generation on the cell surface of membrane extensions ("blebs") 80 to 100 nm in diameter. When competent cells were returned to normal growth conditions, they shed these structures as free vesicles with a concomitant loss of cellular DNA-binding activity. Purified vesicle preparations retained the ability to bind double-stranded DNA in a nuclease-resistant, salt-stable form. Binding was specific for DNA molecules containing the 11-base pair Haemophilus uptake sequence, required Na+ and divalent cations (Mg2+, Ca2+, or Mn2+), and was inhibited by the presence of EDTA or high concentrations of salt (greater than 0.5 M NaCl). Binding was not stimulated by nucleotide triphosphates and was insensitive to the uncoupling agents dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Vesicles contained the major Haemophilus outer membrane proteins and were enriched in several minor proteins.

scholarly journals Designed anchoring geometries determine lifetimes of biotin–streptavidin bonds under constant load and enable ultra-stable coupling

Nanoscale ◽  
2020 ◽  
Vol 12 (41) ◽  
pp. 21131-21137
Author(s):  
Sophia Gruber ◽  
Achim Löf ◽  
Steffen M. Sedlak ◽  
Martin Benoit ◽  
Hermann E. Gaub ◽  
...  
Keyword(s):  
Constant Load ◽  
Magnetic Tweezers ◽  
Constant Force

We engineer streptavidin to control the anchoring geometry and increase the lifetime of the biotin bond under constant force in magnetic tweezers 100-fold.

scholarly journals Conserved Eukaryotic Histone-Fold Residues Substituted into an Archaeal Histone Increase DNA Affinity but Reduce Complex Flexibility

2003 ◽  
Vol 185 (11) ◽  
pp. 3453-3457 ◽  
Author(s):  
Divya J. Soares ◽  
Frédéric Marc ◽  
John N. Reeve
Keyword(s):  
Dna Binding ◽  
Common Ancestor ◽  
Dna Molecules ◽  
Nucleosome Core ◽  
Histone Fold ◽  
Lysine Residues ◽  
Core Histones ◽  
Archaeal Histone

ABSTRACT Although the archaeal and eukaryotic nucleosome core histones evolved from a common ancestor, conserved lysine residues are present at DNA-binding locations in all four eukaryotic histones that are not present in the archaeal histones. Introduction of lysine residues at the corresponding locations into an archaeal histone, HMfB, generated a variant with increased affinity for DNA that formed more compact complexes with DNA. However, these complexes no longer facilitated the circularization of short DNA molecules and had lost the flexibility to wrap DNA alternatively in either a negative or positive supercoil.

scholarly journals Denaturation transition of stretched DNA

2013 ◽  
Vol 41 (2) ◽  
pp. 639-645 ◽  
Author(s):  
Andreas Hanke
Keyword(s):  
Nucleic Acids ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Present Review ◽  
Dna Molecules ◽  
Force Measurements ◽  
Force Extension ◽  
Atomic Force Spectroscopy ◽  
Atomic Force

In the last two decades, single-molecule force measurements using optical and magnetic tweezers and atomic force spectroscopy have dramatically expanded our knowledge of nucleic acids and proteins. These techniques characterize the force on a biomolecule required to produce a given molecular extension. When stretching long DNA molecules, the observed force–extension relationship exhibits a characteristic plateau at approximately 65 pN where the DNA may be extended to almost twice its B-DNA length with almost no increase in force. In the present review, I describe this transition in terms of the Poland–Scheraga model and summarize recent related studies.

Sign in / Sign up

Export Citation Format

Share Document