Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies

Le, Shimin; Yao, Mingxi; Chen, Jin; Efremov, Artem K.; Azimi, Sara; Yan, Jie

doi:10.1093/nar/gkv554

Cited by 38 publications

(42 citation statements)

References 45 publications

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“…An in-house-built vertical magnetic tweezers system ( 42 , 43 ) was used for all single-ssDNA manipulation experiments in this study. A disturbance-free, rapid solution-exchange method using microwell assays ( 44 ) was combined with the magnetic tweezers system to monitor ssDNA extension dynamics during/after protein solution change. The 572-nt ssDNA was prepared in the flow channel as described ( 45 – 47 ).…”

Section: Methodsmentioning

confidence: 99%

Bacillus subtilis RecA with DprA–SsbA antagonizes RecX function during natural transformation

Serrano

Kawamura

et al. 2017

Nucleic Acids Research

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Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA–SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA–SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA–SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA–SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA–SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation.

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Section: Methodsmentioning

confidence: 99%

Bacillus subtilis RecA with DprA–SsbA antagonizes RecX function during natural transformation

Serrano

Kawamura

et al. 2017

Nucleic Acids Research

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“…A vertical magnetic tweezers setup (Chen et al, 2011) was combined with a disturbance-free, rapid solution-exchange flow channel (Le et al, 2015) for protein stretching experiments. A single a-actinin 1 or its sub-domain constructs is tethered between a spycatcher-coated coverslip and a streptavidin-coated paramagnetic bead through N terminus avi-biotin-tag and C terminus spytag.…”

Section: Single-protein Manipulation and Analysismentioning

confidence: 99%

Mechanotransmission and Mechanosensing of Human alpha-Actinin 1

Yao

et al. 2017

Cell Reports

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α-Actinins, a family of critical cytoskeletal actin-binding proteins that usually exist as anti-parallel dimers, play crucial roles in organizing the framework of the cytoskeleton through crosslinking the actin filaments, as well as in focal adhesion maturation. However, the molecular mechanisms underlying its functions are unclear. Here, by mechanical manipulation of single human α-actinin 1 using magnetic tweezers, we determined the mechanical stability and kinetics of the functional domains in α-actinin 1. Moreover, we identified the force-dependence of vinculin binding to α-actinin 1, with the demonstration that force is required to expose the high-affinity binding site for vinculin binding. Further, a role of the α-actinin 1 as molecular shock absorber for the cytoskeleton network is revealed. Our results provide a comprehensive analysis of the force-dependent stability and interactions of α-actinin 1, which sheds important light on the molecular mechanisms underlying its mechanotransmission and mechanosensing functions.

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“…Details of force calibration and force error can be found in our previous publication ( 25 ). A disturbance-free buffer exchange system was used to observe real-time dynamics of DNA extension change ( 27 ). Experimental data included in the main text were carried out at 23°C under the following buffer conditions unless otherwise indicated: 150 mM KCl, 30 mM Tris (pH = 7.4).…”

Section: Methodsmentioning

confidence: 99%

Parallel triplex structure formed between stretched single-stranded DNA and homologous duplex DNA

Chen¹,

Tang²,

Guo³

et al. 2017

Nucleic Acids Research

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The interaction between the single-stranded DNA and the homologous duplex DNA is essential for DNA homologous repair. Here, we report that parallel triplex structure can form spontaneously between a mechanically extended ssDNA and a homologous dsDNA in protein-free condition. The triplex has a contour length close to that of a B-form DNA duplex and remains stable after force is released. The binding energy between the ssDNA and the homologous dsDNA in the triplex is estimated to be comparable to the basepairing energy in a B-form dsDNA. As ssDNA is in a similar extended conformation within recombinase-coated nucleoprotein filaments, we propose that the parallel triplex may form and serve as an intermediate during recombinase-catalyzed homologous joint formation.

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Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies

Cited by 38 publications

References 45 publications

Bacillus subtilis RecA with DprA–SsbA antagonizes RecX function during natural transformation

Bacillus subtilis RecA with DprA–SsbA antagonizes RecX function during natural transformation

Mechanotransmission and Mechanosensing of Human alpha-Actinin 1

Parallel triplex structure formed between stretched single-stranded DNA and homologous duplex DNA

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