scholarly journals Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

2017 ◽  
Vol 13 (2) ◽  
pp. 481-490 ◽  
Author(s):  
Paul B. G. van Erp ◽  
Angela Patterson ◽  
Ravi Kant ◽  
Luke Berry ◽  
Sarah M. Golden ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Dynamics ◽  
Cascade Complex

scholarly journals SUMO-1 regulates the conformational dynamics of Thymine-DNA Glycosylase regulatory domain and competes with its DNA binding activity

2011 ◽  
Vol 12 (1) ◽  
pp. 4 ◽  
Author(s):  
Caroline Smet-Nocca ◽  
Jean-Michel Wieruszeski ◽  
Hélène Léger ◽  
Sebastian Eilebrecht ◽  
Arndt Benecke
Keyword(s):  
Dna Binding ◽  
Conformational Dynamics ◽  
Binding Activity ◽  
Dna Glycosylase ◽  
Regulatory Domain ◽  
Thymine Dna Glycosylase ◽  
Dna Binding Activity

scholarly journals Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

2018 ◽  
Author(s):  
Nilisha Pokhrel ◽  
Colleen C. Caldwell ◽  
Elliot I. Corless ◽  
Emma A. Tillison ◽  
Joseph Tibbs ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Dynamics ◽  
Protein A ◽  
Interacting Proteins ◽  
Ssdna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Mediator Protein ◽  
Damage Response ◽  
The Individual

AbstractReplication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-strand DNA (ssDNA) intermediates, activating the DNA damage response, and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA, but also allow RPA displacement by lower affinity proteins. We have made fluorescent versions of RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA, while RPA as a whole remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. This demonstrates how RPA interacting proteins, with lower ssDNA binding affinity, can access the occluded ssDNA and remodel individual DBDs to replace RPA.One Sentence SummaryThe choreography of binding and rearrangement of the individual domains of RPA during homologous recombination is revealed.

Direct observation of the external force mediated conformational dynamics of an IHF bound Holliday junction

2018 ◽  
Vol 207 ◽  
pp. 251-265
Author(s):  
Subhas C. Bera ◽  
Tapas Paul ◽  
A. N. Sekar Iyengar ◽  
Padmaja P. Mishra
Keyword(s):  
Dna Binding ◽  
External Force ◽  
Single Molecule ◽  
Energy Landscape ◽  
Conformational Dynamics ◽  
Dna Binding Protein ◽  
Integration Host Factor ◽  
Holliday Junctions ◽  
Holliday Junction ◽  
Single Molecule Fret

We have investigated the isomerization dynamics and plausible energy landscape of 4-way Holliday junctions (4WHJs) bound to integration host factor (IHF, a DNA binding protein), considering the effect of applied external force, by single-molecule FRET methods.

scholarly journals Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase

2018 ◽  
Author(s):  
Timothy D. Craggs ◽  
Marko Sustarsic ◽  
Anne Plochowietz ◽  
Majid Mosayebi ◽  
Hendrik Kaju ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Polymerase ◽  
Single Molecule ◽  
Solution Structure ◽  
Binding Proteins ◽  
Conformational Dynamics ◽  
Dna Binding Proteins ◽  
Binary Complex ◽  
Specific Recognition ◽  
Gapped Dna

AbstractDNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120°) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarsegrained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy (<ΔFRET> = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state (ΔG < 4 kT); such conformations were much less accessible to nicked (> 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.Significance StatementMost genetic processes, including DNA replication, repair and transcription, rely on DNA-binding proteins locating specific sites on DNA; some sites contain a specific sequence, whereas others present a specific structure. While sequence-specific recognition has a clear physical basis, structure-specific recognition mechanisms remain obscure. Here, we use single-molecule FRET and computer simulations to show that the conformational dynamics of an important repair intermediate (1nt-gapped DNA) act as central recognition signals for structure-specific binding by DNA polymerase I (Pol). Our conclusion is strongly supported by a novel solution structure of the Pol-DNA complex wherein the gapped-DNA is significantly bent. Our iterative approach combining precise single-molecule measurements with molecular modelling is general and can elucidate the structure and dynamics for many large biomachines.

Effects of electrostatic interactions on global folding and local conformational dynamics of a multidomain Y-family DNA polymerase

2021 ◽  
Author(s):  
Qing-Miao Nie ◽  
Li-Zhen Sun ◽  
Hai-Bin Li ◽  
Xiakun Chu ◽  
Jin Wang
Keyword(s):  
Dna Binding ◽  
Dna Polymerase ◽  
Electrostatic Interactions ◽  
Conformational Dynamics ◽  
Individual Domain ◽  
The Individual ◽  
Y Family

Electrostatic interactions can facilitate the folding of the multidomain DNA polymerase Dpo4 by refining the folding order of the individual domain and promote the functional conformational dynamics of Dpo4 during the DNA-binding recognition.

Conformational dynamics of a transposition repressor in modulating DNA binding

2001 ◽  
Vol 312 (2) ◽  
pp. 311-322 ◽  
Author(s):  
Sadananda S Rai ◽  
Diane O’Handley ◽  
Hiroshi Nakai
Keyword(s):  
Dna Binding ◽  
Conformational Dynamics

scholarly journals DNA binding induces a cis-to-trans switch in Cre recombinase to enable intasome assembly

2020 ◽  
Vol 117 (40) ◽  
pp. 24849-24858
Author(s):  
Aparna Unnikrishnan ◽  
Carlos Amero ◽  
Deepak Kumar Yadav ◽  
Kye Stachowski ◽  
Devante Potter ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Dna Cleavage ◽  
Conformational Dynamics ◽  
Dna Recombination ◽  
Cre Recombinase ◽  
Protein Protein Interactions ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Synaptic Complexes

Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution nuclear magnetic resonance (NMR) spectroscopy to discover the link between intrinsic flexibility and function in Cre recombinase. Transverse relaxation-optimized spectroscopy (TROSY) NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTD and CreCat) to be structurally independent. Amide 15N relaxation measurements of the CreCat domain reveal fast-timescale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein–protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and intermolecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative autoinhibitory conformation for the αN region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site. Moreover, binding to loxP DNA induces a conformational change that dislodges the C terminus, resulting in a cis-to-trans switch that is likely to enable protein–protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a reexamination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.

scholarly journals Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase

2019 ◽  
Vol 47 (20) ◽  
pp. 10788-10800 ◽  
Author(s):  
Timothy D Craggs ◽  
Marko Sustarsic ◽  
Anne Plochowietz ◽  
Majid Mosayebi ◽  
Hendrik Kaju ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Polymerase ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Coarse Grained ◽  
General Mechanism ◽  
Binary Complex ◽  
Specific Recognition ◽  
Gapped Dna ◽  
Dna Substrate

Abstract DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA–Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA–Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4–5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1–2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.

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