scholarly journals DNA Unwinding Mechanism of a RecQ Helicase from Arabidopsis Thaliana Investigated with Magnetic Tweezers

2011 ◽  
Vol 100 (3) ◽  
pp. 23a
Author(s):  
Daniel Klaue ◽  
Daniela Kobbe ◽  
Holger Puchta ◽  
Ralf Seidel
Keyword(s):  
Arabidopsis Thaliana ◽  
Magnetic Tweezers ◽  
Dna Unwinding ◽  
Recq Helicase

scholarly journals Comparing Optical and Magnetic Tweezers for Studying the RecQ Helicase

2014 ◽  
Vol 106 (2) ◽  
pp. 67a
Author(s):  
Maria Marti-Prieto ◽  
Maria Manosas ◽  
Felix Ritort
Keyword(s):  
Magnetic Tweezers ◽  
Recq Helicase

scholarly journals Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

2015 ◽  
Vol 112 (50) ◽  
pp. E6852-E6861 ◽  
Author(s):  
Behzad Rad ◽  
Anthony L. Forget ◽  
Ronald J. Baskin ◽  
Stephen C. Kowalczykowski
Keyword(s):  
Single Molecule ◽  
Fluorescent Sensor ◽  
Holliday Junctions ◽  
Dna Unwinding ◽  
Recq Helicase ◽  
Dna Breaks ◽  
Dna Helicases ◽  
Double Stranded Dna ◽  
Functional Forms ◽  
And Migration

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.

scholarly journals DNA Unwinding by PcrA Helicase and RepD using TIRF and Magnetic Tweezers

2014 ◽  
Vol 106 (2) ◽  
pp. 272a
Author(s):  
Algirdas Toleikis ◽  
Simone Kunzelmann ◽  
Gregory I. Mashanov ◽  
Martin R. Webb ◽  
Justin E. Molloy
Keyword(s):  
Magnetic Tweezers ◽  
Dna Unwinding

scholarly journals Phosphorylation of the RecQ Helicase Sgs1/BLM Controls Its DNA Unwinding Activity during Meiosis and Mitosis

2020 ◽  
Vol 53 (6) ◽  
pp. 706-723.e5 ◽  
Author(s):  
Rokas Grigaitis ◽  
Lepakshi Ranjha ◽  
Philipp Wild ◽  
Kristina Kasaciunaite ◽  
Ilaria Ceppi ◽  
...  
Keyword(s):  
Dna Unwinding ◽  
Recq Helicase

scholarly journals Molecular characteristics of reiterative DNA unwinding by the Caenorhabditis elegans RecQ helicase

2019 ◽  
Vol 47 (18) ◽  
pp. 9708-9720 ◽  
Author(s):  
Seoyun Choi ◽  
Seung-Won Lee ◽  
Hajin Kim ◽  
Byungchan Ahn
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Protein A ◽  
Direct Interaction ◽  
Premature Aging ◽  
Molecular Characteristics ◽  
Dna Unwinding ◽  
Recq Helicase ◽  
Recq Helicases

Abstract The RecQ family of helicases is highly conserved both structurally and functionally from bacteria to humans. Defects in human RecQ helicases are associated with genetic diseases that are characterized by cancer predisposition and/or premature aging. RecQ proteins exhibit 3′-5′ helicase activity and play critical roles in genome maintenance. Recent advances in single-molecule techniques have revealed the reiterative unwinding behavior of RecQ helicases. However, the molecular mechanisms involved in this process remain unclear, with contradicting reports. Here, we characterized the unwinding dynamics of the Caenorhabditis elegans RecQ helicase HIM-6 using single-molecule fluorescence resonance energy transfer measurements. We found that HIM-6 exhibits reiterative DNA unwinding and the length of DNA unwound by the helicase is sharply defined at 25–31 bp. Experiments using various DNA substrates revealed that HIM-6 utilizes the mode of ‘sliding back’ on the translocated strand, without strand-switching for rewinding. Furthermore, we found that Caenorhabditis elegans replication protein A, a single-stranded DNA binding protein, suppresses the reiterative behavior of HIM-6 and induces unidirectional, processive unwinding, possibly through a direct interaction between the proteins. Our findings shed new light on the mechanism of DNA unwinding by RecQ family helicases and their co-operation with RPA in processing DNA.

scholarly journals RecQ helicase triggers a binding mode change in the SSB–DNA complex to efficiently initiate DNA unwinding

2017 ◽  
Vol 45 (20) ◽  
pp. 11878-11890 ◽  
Author(s):  
Maria Mills ◽  
Gábor M. Harami ◽  
Yeonee Seol ◽  
Máté Gyimesi ◽  
Máté Martina ◽  
...  
Keyword(s):  
Binding Mode ◽  
Dna Unwinding ◽  
Recq Helicase ◽  
Mode Change ◽  
Dna Complex

AtRECQ2, a RecQ-helicase homologue from Arabidopsis thaliana, is able to disrupt different recombinogenic DNA-structures in vitro

2008 ◽  
Vol 0 (ja) ◽  
pp. 080414150319983 ◽  
Author(s):  
Daniela Kobbe ◽  
Sandra Blanck ◽  
Katharina Demand ◽  
Manfred Focke ◽  
Holger Puchta
Keyword(s):  
Arabidopsis Thaliana ◽  
Recq Helicase ◽  
Dna Structures

scholarly journals A gate and clamp regulate sequential DNA strand cleavage by CRISPR-Cas12a

2021 ◽  
Author(s):  
Mohsin M Naqvi ◽  
Laura Lee ◽  
Oscar E Torres Montaguth ◽  
Mark Szczelkun
Keyword(s):  
Dna Recognition ◽  
Magnetic Tweezers ◽  
Dna Unwinding ◽  
Nanopore Sequencing ◽  
Loop Formation ◽  
Target Strand ◽  
Diagnostic Applications ◽  
Dna Strand ◽  
Domain Motions ◽  
Resistant State

CRISPR-Cas12a has been widely used for genome editing and diagnostic applications, yet it is not fully understood how RNA-guided DNA recognition activates the sequential cleavage of the non-target strand (NTS) followed by the target strand (TS). Here we used singlemolecule magnetic tweezers microscopy, ensemble gel-based assays and nanopore sequencing to explore the coupling of DNA unwinding and cleavage. In addition to dynamic R-loop formation, we also directly observed transient dsDNA unwinding downstream of the 20 bp DNA:RNA hybrid and, following NTS cleavage and prior to TS cleavage, formation of a hyperstable "clamped" Cas12a-DNA intermediate resistant to DNA twisting. Alanine substitution of a conserved aromatic amino acid "gate" in the REC2 domain that normally caps the heteroduplex produced more frequent and extended downstream DNA breathing, a longer-lived twist-resistant state, and a 16-fold faster rate of TS cleavage. We suggest that both breathing and clamping events, regulated by the gate and by NTS cleavage, deliver the unwound TS to the RuvC nuclease and result from previously described REC2 and NUC domain motions.

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