scholarly journals Decoupling the bridge helix of Cas12a results in a reduced trimming activity and impaired conformational transitions

2021 ◽  
Author(s):  
Elisabeth Wörle ◽  
Leonhard Jakob ◽  
Andreas Schmidbauer ◽  
Gabriel Zinner ◽  
Dina Grohmann
Keyword(s):  
Single Molecule ◽  
Conformational Transitions ◽  
Structural Element ◽  
Francisella Novicida ◽  
Closed State ◽  
Single Molecule Fret ◽  
Type V ◽  
Single Effector ◽  
Cas Proteins

AbstractThe widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by a central structural element, the so-called bridge helix (BH). We generated BH mutants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. Single-molecule FRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH mutants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH mutants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.

scholarly journals Role of the ribosomal protein L27 revealed by single-molecule FRET study

2012 ◽  
Vol 21 (11) ◽  
pp. 1696-1704 ◽  
Author(s):  
Yuhong Wang ◽  
Ming Xiao
Keyword(s):  
Ribosomal Protein ◽  
Single Molecule ◽  
Single Molecule Fret

scholarly journals Conformational transitions in DNA polymerase I revealed by single-molecule FRET

2009 ◽  
Vol 107 (2) ◽  
pp. 715-720 ◽  
Author(s):  
Y. Santoso ◽  
C. M. Joyce ◽  
O. Potapova ◽  
L. Le Reste ◽  
J. Hohlbein ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Single Molecule ◽  
Conformational Transitions ◽  
Dna Polymerase I ◽  
Single Molecule Fret ◽  
Polymerase I

scholarly journals Examining kinesin processivity within a general gating framework

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Johan OL Andreasson ◽  
Bojan Milic ◽  
Geng-Yuan Chen ◽  
Nicholas R Guydosh ◽  
William O Hancock ◽  
...  
Keyword(s):  
Single Molecule ◽  
General Framework ◽  
Optical Trapping ◽  
Atp Hydrolysis ◽  
Wild Type ◽  
Structural Element ◽  
Gating Mechanisms ◽  
Motor Velocity

Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ∼1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.

scholarly journals Single Molecule FRET Studies of RNA Tertiary Folding: Elucidating the Thermodynamic and Kinetic Role of Na+ Cations

2010 ◽  
Vol 98 (3) ◽  
pp. 471a
Author(s):  
Erik D. Holmstrom ◽  
Julie L. Fiore ◽  
David Nesbitt
Keyword(s):  
Single Molecule ◽  
Single Molecule Fret ◽  
Tertiary Folding

scholarly journals Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy

RNA ◽  
2013 ◽  
Vol 19 (4) ◽  
pp. 517-526 ◽  
Author(s):  
N. Beerens ◽  
M. D. E. Jepsen ◽  
V. Nechyporuk-Zloy ◽  
A. C. Kruger ◽  
J.-L. Darlix ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Molecule Fret ◽  
Hiv 1

scholarly journals ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Su Hyung Park ◽  
Nalae Kang ◽  
Eunho Song ◽  
Minwoo Wie ◽  
Eun A. Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Cultured Cells ◽  
Replication Stress ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Single Molecule Fret ◽  
Hydroxyurea Treatment ◽  
Fork Regression

AbstractMaintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2ʹ-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.

scholarly journals Conformational Transitions in the Glycine-Bound GluN1 NMDA Receptor LBD via Single-Molecule FRET

2015 ◽  
Vol 109 (1) ◽  
pp. 66-75 ◽  
Author(s):  
David R. Cooper ◽  
Drew M. Dolino ◽  
Henriette Jaurich ◽  
Bo Shuang ◽  
Swarna Ramaswamy ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Single Molecule ◽  
Conformational Transitions ◽  
Single Molecule Fret

scholarly journals Real-time observation of flexible domain movements in Cas9

2017 ◽  
Author(s):  
Saki Osuka ◽  
Kazushi Isomura ◽  
Shohei Kajimoto ◽  
Tomotaka Komori ◽  
Hiroshi Nishimasu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Potential Role ◽  
Domain Architecture ◽  
Guide Rna ◽  
Single Molecule Fret ◽  
Target Dna ◽  
Time Observation ◽  
Real Time Observation

ABSTRACTThe CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains, but whether these flexible movements occur in solution is unclear. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET. The flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain in Cas9 only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.

Sign in / Sign up

Export Citation Format

Share Document