scholarly journals Single-molecule analysis of RAG-mediated V(D)J DNA cleavage

2015 ◽  
Vol 112 (14) ◽  
pp. E1715-E1723 ◽  
Author(s):  
Geoffrey A. Lovely ◽  
Robert C. Brewster ◽  
David G. Schatz ◽  
David Baltimore ◽  
Rob Phillips
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Synapse Formation ◽  
High Mobility ◽  
Binding Energies ◽  
Synaptic Complex ◽  
Single Molecule Level ◽  
Molecular Events ◽  
The Individual ◽  
Single Molecule Analysis

The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed single-molecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only ∼40% of observed synapses resulting in cleavage at consensus RSS binding sites.

scholarly journals Real-time analysis of RAG complex activity in V(D)J recombination

2016 ◽  
Vol 113 (42) ◽  
pp. 11853-11858 ◽  
Author(s):  
Jennifer Zagelbaum ◽  
Noriko Shimazaki ◽  
Zitadel Anne Esguerra ◽  
Go Watanabe ◽  
Michael R. Lieber ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Conformational Changes ◽  
Signal Sequence ◽  
High Mobility ◽  
Time Analysis ◽  
Complex Activity ◽  
Synaptic Complex ◽  
Single Molecule Fret ◽  
Real Time Analysis

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS–RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

scholarly journals Local, Single-Molecule Oxidative Cleaving of DNA Origami by C60 on an AFM Tip

2021 ◽  
Author(s):  
Ankita Ray ◽  
Cristiana Passiu ◽  
S.N Ramakrishna ◽  
Antonella Rossi ◽  
Akinori Kuzuya ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Oxidative Damage ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Dna Origami ◽  
Oxygen Species ◽  
Mica Surface ◽  
Single Molecule Level ◽  
Afm Tips ◽  
Morphology Changes

Spatially controlled single-molecule oxidation of DNA was performed by photocatalytic generation of singlet oxygen on chemically functionalized AFM tips. A waffle-type DNA origami deposited on a mica surface is site-specifically destroyed by generation of reactive oxygen species at the AFM tip, which was modified with C<sub>60</sub>-tripod photocatalyst. Upon AFM scanning under photoirradiation, DNA morphology changes, corresponding to oxidative damage were clearly observed at the single-molecule level. The DNA cleavage occurred with strict dependence on photoirradiation and the presence of C<sub>60 </sub>on the AFM tip.

scholarly journals Transition from stochastic events to deterministic ensemble average in electron transfer reactions revealed by single-molecule conductance measurement

2019 ◽  
Vol 116 (9) ◽  
pp. 3407-3412 ◽  
Author(s):  
Yueqi Li ◽  
Hui Wang ◽  
Zixiao Wang ◽  
Yanjun Qiao ◽  
Jens Ulstrup ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Single Molecule ◽  
Electron Transfer Reaction ◽  
Transfer Reactions ◽  
Ensemble Averaging ◽  
Electron Transfer Reactions ◽  
Single Molecule Level ◽  
Individual Reaction ◽  
Stochastic Events ◽  
The Individual

Electron transfer reactions can now be followed at the single-molecule level, but the connection between the microscopic and macroscopic data remains to be understood. By monitoring the conductance of a single molecule, we show that the individual electron transfer reaction events are stochastic and manifested as large conductance fluctuations. The fluctuation probability follows first-order kinetics with potential dependent rate constants described by the Butler–Volmer relation. Ensemble averaging of many individual reaction events leads to a deterministic dependence of the conductance on the external electrochemical potential that follows the Nernst equation. This study discloses a systematic transition from stochastic kinetics of individual reaction events to deterministic thermodynamics of ensemble averages and provides insights into electron transfer processes of small systems, consisting of a single molecule or a small number of molecules.

scholarly journals α-Actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk

2017 ◽  
Vol 114 (5) ◽  
pp. 1015-1020 ◽  
Author(s):  
Marco Grison ◽  
Ulrich Merkel ◽  
Julius Kostan ◽  
Kristina Djinović-Carugo ◽  
Matthias Rief
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Kinetic Properties ◽  
Single Molecule Level ◽  
Passive Stretching ◽  
Dual Beam ◽  
Important Interaction ◽  
Cross Linker ◽  
The Individual

Stable anchoring of titin within the muscle Z-disk is essential for preserving muscle integrity during passive stretching. One of the main candidates for anchoring titin in the Z-disk is the actin cross-linker α-actinin. The calmodulin-like domain of α-actinin binds to the Z-repeats of titin. However, the mechanical and kinetic properties of this important interaction are still unknown. Here, we use a dual-beam optical tweezers assay to study the mechanics of this interaction at the single-molecule level. A single interaction of α-actinin and titin turns out to be surprisingly weak if force is applied. Depending on the direction of force application, the unbinding forces can more than triple. Our results suggest a model where multiple α-actinin/Z-repeat interactions cooperate to ensure long-term stable titin anchoring while allowing the individual components to exchange dynamically.

Single-molecule analysis of the self-assembly process facilitated by host–guest interactions

2015 ◽  
Vol 51 (7) ◽  
pp. 1202-1205 ◽  
Author(s):  
Fu-Na Meng ◽  
Xuyang Yao ◽  
Yi-Lun Ying ◽  
Junji Zhang ◽  
He Tian ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Methyl Viologen ◽  
The Self ◽  
Assembly Process ◽  
Single Molecule Level ◽  
Single Molecule Analysis

The self-assembly process operated by para-sulfonatocalix[6]arenes and methyl viologen was analyzed at the single-molecule level through an α-hemolysin nanopore.

scholarly journals Local, Single-Molecule Oxidative Cleaving of DNA Origami by C60 on an AFM Tip

2021 ◽  
Author(s):  
Ankita Ray ◽  
Cristiana Passiu ◽  
S.N Ramakrishna ◽  
Antonella Rossi ◽  
Akinori Kuzuya ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Oxidative Damage ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Dna Origami ◽  
Oxygen Species ◽  
Mica Surface ◽  
Single Molecule Level ◽  
Afm Tips ◽  
Morphology Changes

Spatially controlled single-molecule oxidation of DNA was performed by photocatalytic generation of singlet oxygen on chemically functionalized AFM tips. A waffle-type DNA origami deposited on a mica surface is site-specifically destroyed by generation of reactive oxygen species at the AFM tip, which was modified with C<sub>60</sub>-tripod photocatalyst. Upon AFM scanning under photoirradiation, DNA morphology changes, corresponding to oxidative damage were clearly observed at the single-molecule level. The DNA cleavage occurred with strict dependence on photoirradiation and the presence of C<sub>60 </sub>on the AFM tip.

scholarly journals Programming PAM antennae for efficient CRISPR-Cas9 DNA editing

2020 ◽  
Vol 6 (19) ◽  
pp. eaay9948
Author(s):  
Fei Wang ◽  
Yaya Hao ◽  
Qian Li ◽  
Jiang Li ◽  
Honglu Zhang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Super Resolution ◽  
Single Molecule Level ◽  
Guide Rna ◽  
Guide Rnas ◽  
Protospacer Adjacent Motif ◽  
Target Dna

Bacterial CRISPR-Cas9 nucleases have been repurposed as powerful genome editing tools. Whereas engineering guide RNAs or Cas nucleases have proven to improve the efficiency of CRISPR editing, modulation of protospacer-adjacent motif (PAM), indispensable for CRISPR, has been less explored. Here, we develop a DNA origami–based platform to program a PAM antenna microenvironment and address its performance at the single-molecule level with submolecular resolution. To mimic spatially controlled in vivo PAM distribution as may occur in chromatin, we investigate the effect of PAM antennae surrounding target DNA. We find that PAM antennae effectively sensitize the DNA cleavage by recruiting Cas9 molecules. Super-resolution tracking of single single-guide RNA/Cas9s reveals localized translocation of Cas9 among spatially proximal PAMs. We find that the introduction of the PAM antennae effectively modulates the microenvironment for enhanced target cleavage (up to ~50%). These results provide insight into factors that promote more efficient genome editing.

scholarly journals Mapping DNA replication with nanopore sequencing

2018 ◽  
Author(s):  
Magali Hennion ◽  
Jean-Michel Arbona ◽  
Corinne Cruaud ◽  
Florence Proux ◽  
Benoît Le Tallec ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Electrical Signal ◽  
Yeast Cells ◽  
Nanopore Sequencing ◽  
Single Molecule Level ◽  
Experimental Systems ◽  
Genome Wide ◽  
Single Molecule Analysis

ABSTRACTWe have harnessed nanopore sequencing to study DNA replication genome-wide at the single-molecule level. Using in vitro prepared DNA substrates, we characterized the effect of bromodeoxyuridine (BrdU) substitution for thymidine on the MinION nanopore electrical signal. Using a neural-network basecaller trained on yeast DNA containing either BrdU or thymidine, we identified BrdU-labelled tracts in yeast cells synchronously entering S phase in the presence of hydroxyurea and BrdU. As expected, the BrdU-labelled tracts coincided with previously identified early-firing, but not late-firing, replication origins. These results open the way to high-throughput, high-resolution, single-molecule analysis of DNA replication in many experimental systems.

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