scholarly journals H2A.Z deposition by SWR1C involves multiple ATP-dependent steps

2022 ◽  
Author(s):  
Jiayi Fan ◽  
Andrew T Moreno ◽  
Alexander S Baier ◽  
Joseph J Loparo ◽  
Craig L Peterson
Keyword(s):  
Single Molecule ◽  
Reaction Pathway ◽  
Histone H2a ◽  
Time Analysis ◽  
Rapid Loss ◽  
Protein Coding ◽  
Nucleosome Remodeling ◽  
Real Time Analysis ◽  
Dna Unwrapping ◽  
Genomic Locations

The histone variant H2A.Z is a conserved feature of nucleosomes flanking protein-coding genes. Deposition of H2A.Z requires ATP-dependent replacement of nucleosomal H2A by a chromatin remodeler related to the multi-subunit enzyme, yeast SWR1C. How these enzymes use ATP to promote this nucleosome editing reaction remains unclear. Here we use single-molecule and ensemble methodologies to identify three ATP-dependent phases in the H2A.Z deposition reaction. Real-time analysis of single nucleosome remodeling events reveals an initial, priming step that occurs after ATP addition that likely involves transient DNA unwrapping from the nucleosome. Priming is followed by rapid loss of histone H2A, which is subsequently released from the H2A.Z nucleosomal product. Surprisingly, the rates of both priming and the release of the H2A/H2B dimer are sensitive to ATP concentration. This complex reaction pathway provides multiple opportunities to regulate the timely and accurate deposition of H2A.Z at key genomic locations.

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 Real-Time Analysis and Visualization for Single-Molecule Based Super-Resolution Microscopy

PLoS ONE ◽  
2013 ◽  
Vol 8 (4) ◽  
pp. e62918 ◽  
Author(s):  
Adel Kechkar ◽  
Deepak Nair ◽  
Mike Heilemann ◽  
Daniel Choquet ◽  
Jean-Baptiste Sibarita
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Super Resolution ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Super Resolution Microscopy

scholarly journals Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel

2012 ◽  
Vol 109 (22) ◽  
pp. 8477-8482 ◽  
Author(s):  
B. R. Cipriany ◽  
P. J. Murphy ◽  
J. A. Hagarman ◽  
A. Cerf ◽  
D. Latulippe ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Time Analysis ◽  
Methylated Dna ◽  
Real Time Analysis ◽  
Selection Of

scholarly journals Yeast Chd1p remodels nucleosomes with unique DNA unwrapping and translocation dynamics

2018 ◽  
Author(s):  
Jaewon Kirk ◽  
Ju Yeon Lee ◽  
Yejin Lee ◽  
Chanshin Kang ◽  
Soochul Shin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Chromatin Remodeler ◽  
Base Pairs ◽  
Nucleosome Remodeling ◽  
Chromatin Remodelers ◽  
Histone Octamer ◽  
Single Molecule Fret ◽  
Single Molecule Studies ◽  
Dna Unwrapping

AbstractChromodomain-helicase-DNA-binding protein 1 (CHD1) remodels chromatin by translocating nucleosomes along DNA, but its mechanism remains poorly understood. Here, we employ a single-molecule fluorescence approach to characterize nucleosome remodeling by yeast CHD1 (Chd1p). We show that Chd1p translocates nucleosomes in steps of multiple base pairs per ATP. ATP binding to Chd1p induces a transient unwrapping of the exit-side DNA, and facilitates nucleosome translocation. ATP hydrolysis induces nucleosome translocation, which is followed by the rewrapping upon the release of the hydrolyzed nucleotide. Multiple Chd1ps binding to a single nucleosome sequentially moves a histone octamer with a preference to the center of DNA fragments, suggesting a new mechanism for regularly spaced nucleosome generation by Chd1p. Our results reveal the unique mechanism by which Chd1p remodels nucleosomes.Significance StatementThere are four major ATP-dependent chromatin remodeler families: SWI/SNF, ISWI, CHD, and INO80/SWR1. The remodeling mechanisms of SWI/SNF and ISWI chromatin remodelers have been elucidated through extensive single-molecule studies, but it remains poorly understood how CHD chromatin remodeler operate. We use single-molecule FRET techniques, and show that Yeast CHD1 uses unique mechanisms to remodel a nucleosome.

An Interactive Minicomputer Program for the Indexing and Simulation of Electron Diffraction Patterns

1976 ◽  
Vol 34 ◽  
pp. 542-543
Author(s):  
R.P. Goehner ◽  
W.T. Hatfield ◽  
Prakash Rao
Keyword(s):  
Electron Diffraction ◽  
Real Time ◽  
Pattern Analysis ◽  
Flow Diagram ◽  
Hard Copy ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Transmission Electron ◽  
One Step ◽  
Diffraction Patterns

Computer programs are now available in various laboratories for the indexing and simulation of transmission electron diffraction patterns. Although these programs address themselves to the solution of various aspects of the indexing and simulation process, the ultimate goal is to perform real time diffraction pattern analysis directly off of the imaging screen of the transmission electron microscope. The program to be described in this paper represents one step prior to real time analysis. It involves the combination of two programs, described in an earlier paper(l), into a single program for use on an interactive basis with a minicomputer. In our case, the minicomputer is an INTERDATA 70 equipped with a Tektronix 4010-1 graphical display terminal and hard copy unit.A simplified flow diagram of the combined program, written in Fortran IV, is shown in Figure 1. It consists of two programs INDEX and TEDP which index and simulate electron diffraction patterns respectively. The user has the option of choosing either the indexing or simulating aspects of the combined program.

scholarly journals Real-Time Analysis of Animal Feeding Behavior With a Low-Calculation-Power CPU

2020 ◽  
Vol 67 (4) ◽  
pp. 1197-1205 ◽  
Author(s):  
Yuki Totani ◽  
Susumu Kotani ◽  
Kei Odai ◽  
Etsuro Ito ◽  
Manabu Sakakibara
Keyword(s):  
Feeding Behavior ◽  
Real Time ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Animal Feeding
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