Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

10.3791/62112 ◽  
2021 ◽  
Author(s):  
Candice M. Etson ◽  
Petar Todorov ◽  
Nooshin Shatery Nejad ◽  
Nirmala Shrestha ◽  
David R. Walt
Keyword(s):  
Restriction Endonuclease ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Dwell Time ◽  
Time Analysis

Single-Molecule Force-Clamp Spectroscopy: Dwell Time Analysis and Practical Considerations†

Langmuir ◽  
2011 ◽  
Vol 27 (4) ◽  
pp. 1440-1447 ◽  
Author(s):  
Yi Cao ◽  
Hongbin Li
Keyword(s):  
Single Molecule ◽  
Dwell Time ◽  
Time Analysis ◽  
Force Clamp

Dwell Time Analysis of a Single-Molecule Mechanochemical Reaction†

Langmuir ◽  
2008 ◽  
Vol 24 (4) ◽  
pp. 1356-1364 ◽  
Author(s):  
Robert Szoszkiewicz ◽  
Sri Rama Koti Ainavarapu ◽  
Arun P. Wiita ◽  
Raul Perez-Jimenez ◽  
Jose M. Sanchez-Ruiz ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dwell Time ◽  
Mechanochemical Reaction ◽  
Time Analysis

scholarly journals Power-law behavior of transcription factor dynamics at the single-molecule level implies a continuum affinity model

2021 ◽  
Author(s):  
David A Garcia ◽  
Gregory Fettweis ◽  
Diego M Presman ◽  
Ville Paakinaho ◽  
Christopher Jarzynski ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Power Law ◽  
Dwell Time ◽  
Specific Binding ◽  
Power Law Distribution ◽  
Single Molecule Level ◽  
Binding Behavior ◽  
Chromatin Template ◽  
Nuclear Domains

Abstract Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs—one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template.

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.

How Powerful is the Dwell-Time Analysis of Multichannel Records?

1998 ◽  
Vol 165 (1) ◽  
pp. 19-35 ◽  
Author(s):  
R. Blunck ◽  
U. Kirst ◽  
T. Riessner ◽  
U.-P. Hansen
Keyword(s):  
Dwell Time ◽  
Time Analysis

Stability Analysis for a Class of Linear Switched Positive Systems

2012 ◽  
Vol 562-564 ◽  
pp. 2084-2087
Author(s):  
Hui Ding ◽  
Xu Yang Lou
Keyword(s):  
Lyapunov Functions ◽  
Dwell Time ◽  
Positive Systems ◽  
Time Analysis ◽  
Arbitrary Switching ◽  
The Family ◽  
The Common ◽  
The Stability ◽  
Switched Positive Systems ◽  
Explicit Relation

This paper addresses stability properties of linear switched positive systems composed of continuous-time subsystems and discrete-time subsystems. Based on the common linear copositive Lyapunov functions, stability of the positive systems is discussed under arbitrary switching. Moreover, a sufficient condition on the minimum dwell time that guarantees the stability of linear switched positive systems. The dwell time analysis interprets the stability of linear switched positive systems through the distance between the eigenvector sets. Thus, an explicit relation in view of stability is obtained between the family of the involved subsystems and the set of admissible switching signals.

scholarly journals Activation-induced Deaminase (AID)-directed Hypermutation in the Immunoglobulin Sμ Region

2002 ◽  
Vol 195 (4) ◽  
pp. 529-534 ◽  
Author(s):  
Hitoshi Nagaoka ◽  
Masamichi Muramatsu ◽  
Namiko Yamamura ◽  
Kazuo Kinoshita ◽  
Tasuku Honjo
Keyword(s):  
B Lymphocytes ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Somatic Hypermutation ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Variable Regions ◽  
Class Switch ◽  
Large Deletions ◽  
Activation Induced Deaminase

Somatic hypermutation (SHM) and class switch recombination (CSR) cause distinct genetic alterations at different regions of immunoglobulin genes in B lymphocytes: point mutations in variable regions and large deletions in S regions, respectively. Yet both depend on activation-induced deaminase (AID), the function of which in the two reactions has been an enigma. Here we report that B cell stimulation which induces CSR but not SHM, leads to AID-dependent accumulation of SHM-like point mutations in the switch μ region, uncoupled with CSR. These findings strongly suggest that AID itself or a single molecule generated by RNA editing function of AID may mediate a common step of SHM and CSR, which is likely to be involved in DNA cleavage.

scholarly journals Using Single Molecule Methods to Study Mechanisms of Site Specific DNA Cleavage

2019 ◽  
Vol 116 (3) ◽  
pp. 504a
Author(s):  
Allen C. Price ◽  
Stephen D. Parziale ◽  
Karissa Mehrtens ◽  
Anna D. Ware ◽  
Emily K. Matozel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Site Specific
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