scholarly journals Studies of bacterial topoisomerases I and III at the single-molecule level

2013 ◽  
Vol 41 (2) ◽  
pp. 571-575 ◽  
Author(s):  
Ksenia Terekhova ◽  
John F. Marko ◽  
Alfonso Mondragón
Keyword(s):  
Single Molecule ◽  
Topoisomerase I ◽  
Dna Topology ◽  
Biological Molecules ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Topoisomerase Iii ◽  
Type Ia ◽  
Dna Strand

Topoisomerases are the enzymes responsible for maintaining the supercoiled state of DNA in the cell and also for many other DNA-topology-associated reactions. Type IA enzymes alter DNA topology by breaking one DNA strand and passing another strand or strands through the break. Although all type IA topoisomerases are related at the sequence, structure and mechanism levels, different type IA enzymes do not participate in the same cellular processes. We have studied the mechanism of DNA relaxation by Escherichia coli topoisomerases I and III using single-molecule techniques to understand their dissimilarities. Our experiments show important differences at the single-molecule level, while also recovering the results from bulk experiments. Overall, topoisomerase III relaxes DNA using fast processive runs followed by long pauses, whereas topoisomerase I relaxes DNA through slow processive runs followed by short pauses. These two properties combined give rise to the overall relaxation rate, which is higher for topoisomerase I than for topoisomerase III, as expected from many biochemical observations. The results help us to understand better the role of these two topoisomerases in the cell and also serve to illustrate the power of single-molecule experiments to uncover new functional characteristics of biological molecules.

Curaxin-Induced DNA Topology Alterations Trigger the Distinct Binding Response of CTCF and FACT at the Single-Molecule Level

Biochemistry ◽  
2021 ◽  
Vol 60 (7) ◽  
pp. 494-499
Author(s):  
Ke Lu ◽  
Cuifang Liu ◽  
Yinuo Liu ◽  
Anfeng Luo ◽  
Jun Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Topology ◽  
Single Molecule Level

scholarly journals A highly processive topoisomerase I: studies at the single-molecule level

2014 ◽  
Vol 42 (12) ◽  
pp. 7935-7946 ◽  
Author(s):  
Marcin Jan Szafran ◽  
Terence Strick ◽  
Agnieszka Strzałka ◽  
Jolanta Zakrzewska-Czerwińska ◽  
Dagmara Jakimowicz
Keyword(s):  
Single Molecule ◽  
Topoisomerase I ◽  
Single Molecule Level

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

2020 ◽  
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
High Probability ◽  
Actin Binding ◽  
Single Molecule Level ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

Nanomechanochemical Delivery of Nanoparticles for Nanomechanics Inside Living Cells

2010 ◽  
Author(s):  
Kyungsuk Yum ◽  
Sungsoo Na ◽  
Yang Xiang ◽  
Ning Wang ◽  
Min-Feng Yu
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Endocytic Pathway ◽  
Great Promise ◽  
Biological Processes ◽  
Temporal Precision ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Biological Studies

Studying biological processes and mechanics in living cells is challenging but highly rewarding. Recent advances in experimental techniques have provided numerous ways to investigate cellular processes and mechanics of living cells. However, most of existing techniques for biomechanics are limited to experiments outside or on the membrane of cells, due to the difficulties in physically accessing the interior of living cells. On the other hand, nanomaterials, such as fluorescent quantum dots (QDs) and magnetic nanoparticles, have shown great promise to overcome such limitations due to their small sizes and excellent functionalities, including bright and stable fluorescence and remote manipulability. However, except a few systems, the use of nanoparticles has been limited to the study of biological studies on cell membranes or related to endocytosis, because of the difficulty of delivering dispersed and single nanoparticles into living cells. Various strategies have been explored, but delivered nanoparticles are often trapped in the endocytic pathway or form aggregates in the cytoplasm, limiting their further use. Here we show a nanoscale direct delivery method, named nanomechanochemical delivery, where we manipulate a nanotube-based nanoneedle, carrying “cargo” (QDs in this study), to mechanically penetrate the cell membrane, access specific areas inside cells, and release the cargo [1]. We selectively delivered well-dispersed QDs into either the cytoplasm or the nucleus of living cells. We quantified the dynamics of the delivered QDs by single-molecule tracking and demonstrated the applicability of the QDs as a nanoscale probe for studying nanomechanics inside living cells (by using the biomicrorhology method), revealing the biomechanical heterogeneity of the cellular environment. This method may allow new strategies for studying biological processes and mechanics in living cells with spatial and temporal precision, potentially at the single-molecule level.

scholarly journals Affinity of Skp to OmpC revealed by single-molecule detection

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sichen Pan ◽  
Chen Yang ◽  
Xin Sheng Zhao
Keyword(s):  
Single Molecule ◽  
Molecular Chaperones ◽  
Outer Membrane Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Hill Coefficient ◽  
Stoichiometric Ratio ◽  
Single Molecule Level

Abstract Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 μM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.

scholarly journals Topoisomerase I as a Biomarker: Detection of Activity at the Single Molecule Level

Sensors ◽  
2014 ◽  
Vol 14 (1) ◽  
pp. 1195-1207 ◽  
Author(s):  
Joanna Proszek ◽  
Amit Roy ◽  
Ann-Katrine Jakobsen ◽  
Rikke Frøhlich ◽  
Birgitta Knudsen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Topoisomerase I ◽  
Biomarker Detection ◽  
Single Molecule Level

scholarly journals DNA Repair Functions That Control Sensitivity to Topoisomerase-Targeting Drugs

2004 ◽  
Vol 3 (1) ◽  
pp. 82-90 ◽  
Author(s):  
Mobeen Malik ◽  
John L. Nitiss
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Topoisomerase I ◽  
Topoisomerase Ii ◽  
Excision Repair ◽  
Dna Strand Breaks ◽  
Dna Topology ◽  
Strand Breaks ◽  
Wide Range ◽  
Dna Strand

ABSTRACT DNA topoisomerases play critical roles in a wide range of cellular processes by altering DNA topology to facilitate replication, transcription, and chromosome segregation. Topoisomerases alter DNA topology by introducing transient DNA strand breaks that involve a covalent protein DNA intermediate. Many agents have been found to prevent the religation of DNA strand breaks induced by the enzymes, thereby converting the enzymes into DNA-damaging agents. Repair of the DNA damage induced by topoisomerases is significant in understanding drug resistance arising following treatment with topoisomerase-targeting drugs. We have used the fission yeast Schizosaccharomyces pombe to identify DNA repair pathways that are important for cell survival following drug treatment. S. pombe strains carrying mutations in genes required for homologous recombination such as rad22A or rad32 (homologues of RAD52 and MRE11) are hypersensitive to drugs targeting either topoisomerase I or topoisomerase II. In contrast to results observed with Saccharomyces cerevisiae, S. pombe strains defective in nucleotide excision repair are also hypersensitive to topoisomerase-targeting agents. The loss of DNA replication or DNA damage checkpoints also sensitizes cells to both topoisomerase I and topoisomerase II inhibitors. Finally, repair genes (such as the S. pombe rad8+ gene) with no obvious homologs in other systems also play important roles in causing sensitivity to topoisomerase drugs. Since the pattern of sensitivity is distinct from that seen with other systems (such as the S. cerevisiae system), our results highlight the usefulness of S. pombe in understanding how cells deal with the unique DNA damage induced by topoisomerases.

PRINCIPLES OF SINGLE-MOLECULE MANIPULATION AND ITS APPLICATION IN BIOLOGICAL PHYSICS

2012 ◽  
Vol 26 (13) ◽  
pp. 1230006 ◽  
Author(s):  
WEI-HUNG CHEN ◽  
JONATHAN D. WILSON ◽  
SITHARA S. WIJERATNE ◽  
SARAH A. SOUTHMAYD ◽  
KUAN-JIUH LIN ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Biological Molecules ◽  
Force Detection ◽  
Biomolecular Systems ◽  
Single Molecule Level ◽  
Detection Techniques ◽  
Atomic Force ◽  
Manipulation System ◽  
Single Molecule Manipulation

Recent advances in nanoscale manipulation and piconewton force detection provide a unique tool for studying the mechanical and thermodynamic properties of biological molecules and complexes at the single-molecule level. Detailed equilibrium and dynamics information on proteins and DNA have been revealed by single-molecule manipulation and force detection techniques. The atomic force microscope (AFM) and optical tweezers have been widely used to quantify the intra- and inter-molecular interactions of many complex biomolecular systems. In this article, we describe the background, analysis, and applications of these novel techniques. Experimental procedures that can serve as a guide for setting up a single-molecule manipulation system using the AFM are also presented.

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