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Journal ArticleDOI

Fork sensing and strand switching control antagonistic activities of RecQ helicases

TL;DR: This work investigates the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECZ3 at the single-molecule level using magnetic tweezers and provides a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family.
Abstract: RecQ helicases have essential roles in maintaining genome stability during replication and in controlling double-strand break repair by homologous recombination. Little is known about how the different RecQ helicases found in higher eukaryotes achieve their specialized and partially opposing functions. Here, we investigate the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECQ3 at the single-molecule level using magnetic tweezers. Although AtRECQ2 predominantly unwinds forked DNA substrates in a highly repetitive fashion, AtRECQ3 prefers to rewind, that is, to close preopened DNA forks. For both enzymes, this process is controlled by frequent strand switches and active sensing of the unwinding fork. The relative extent of the strand switches towards unwinding or towards rewinding determines the predominant direction of the enzyme. Our results provide a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family.

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Citations
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Journal ArticleDOI
TL;DR: This work demonstrates that camera-based imaging can provide a similar performance for all three dimensions of particle tracking with Ångström accuracy as laser detection through photodiodes, and provides a simple and robust way for high-resolution tweezers experiments using multiple particles at a time.
Abstract: Particle tracking with ultra-high resolution in optical and magnetic tweezers has so far relied on laser detection through photodiodes. Here, Huhle et al. demonstrate three-dimensional particle tracking with Angstrom accuracy and real-time GPU-accelerated data processing at kHz rates using camera-based imaging.

113 citations

Journal ArticleDOI
TL;DR: It is shown that the activity of BLM is substrate dependent, and highly regulated by a short ssDNA segment that separates the G4 motif from dsDNA, and a model is presented that proposes a unique role for G4 structures in modulating theActivity of DNA processing enzymes.
Abstract: Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA strands and play an important role in the maintenance of genomic integrity. Here we used single-molecule FRET to define the mechanism of interaction of BLM helicase with intra-stranded G4 structures. We show that the activity of BLM is substrate dependent, and highly regulated by a short-strand DNA (ssDNA) segment that separates the G4 motif from double-stranded DNA. We demonstrate cooperativity between the RQC and HRDC domains of BLM during binding and unfolding of the G4 structure, where the RQC domain interaction with G4 is stabilized by HRDC binding to ssDNA. We present a model that proposes a unique role for G4 structures in modulating the activity of DNA processing enzymes.

80 citations

Journal ArticleDOI
09 Sep 2016-eLife
TL;DR: It is shown that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine, which collectively suggests that the h DNA2 motor promotes the enzyme's capacity to degrade ds DNA in conjunction with BLMor WRN and thus promote the repair of broken DNA.
Abstract: Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.

64 citations


Cites background from "Fork sensing and strand switching c..."

  • ...In agreement with this no direction reversals during unwinding (i.e. rezipping) that could originate from strand–switches were observed (Dessinges et al., 2004; Klaue et al., 2013)....

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  • ...rezipping) that could originate from strand–switches were observed (Dessinges et al., 2004; Klaue et al., 2013)....

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Journal ArticleDOI
TL;DR: The findings reveal that E1 employs a strand exclusion mechanism to unwind DNA with the N-terminal side leading at the replication fork, and DNA unwinding by E1 is modulated by the origin-recognition domain, suggesting a previously unsuspected role for this domain in regulating helicase activity.
Abstract: A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms.

61 citations

Journal ArticleDOI
TL;DR: The force calibration based on the long pendulum geometry will facilitate high-resolution magnetic-tweezers experiments that rely on short molecules and large forces, as well as highly parallelized measurements that use low frame rates.

56 citations

References
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Journal ArticleDOI
TL;DR: This review sets out to define a nomenclature for helicase and translocase enzymes based on current knowledge of sequence, structure, and mechanism, and delineate six superfamilies of enzymes, with examples of crystal structures where available.
Abstract: Helicases and translocases are a ubiquitous, highly diverse group of proteins that perform an extraordinary variety of functions in cells. Consequently, this review sets out to define a nomenclature for these enzymes based on current knowledge of sequence, structure, and mechanism. Using previous definitions of helicase families as a basis, we delineate six superfamilies of enzymes, with examples of crystal structures where available, and discuss these structures in the context of biochemical data to outline our present understanding of helicase and translocase activity. As a result, each superfamily is subdivided, where appropriate, on the basis of mechanistic understanding, which we hope will provide a framework for classification of new superfamily members as they are discovered and characterized.

1,145 citations

Journal ArticleDOI
02 Apr 1999-Cell
TL;DR: Two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex are determined, providing snapshots of different steps on the catalytic pathway, providing evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism.

759 citations

Journal ArticleDOI
TL;DR: The structure of the HCV NS3 RNA helicase domain complexed with a single-stranded DNA oligonucleotide has been solved to 2.2 A resolution and is a member of a superfamily of helicases, termed superfamily II.

630 citations

Journal ArticleDOI
TL;DR: This Review discusses how these proteins might suppress genomic rearrangements, and therefore function as 'caretaker' tumour suppressors.
Abstract: Around 1% of the open reading frames in the human genome encode predicted DNA and RNA helicases. One highly conserved group of DNA helicases is the RecQ family. Genetic defects in three of the five human RecQ helicases, BLM, WRN and RECQ4, give rise to defined syndromes associated with cancer predisposition, some features of premature ageing and chromosomal instability. In recent years, there has been a tremendous advance in our understanding of the cellular functions of individual RecQ helicases. In this Review, we discuss how these proteins might suppress genomic rearrangements, and therefore function as 'caretaker' tumour suppressors.

432 citations

Journal ArticleDOI
29 Dec 2006-Cell
TL;DR: A series of crystal structures of the UvrD helicase complexed with DNA and ATP hydrolysis intermediates reveal that ATP binding alone leads to unwinding of 1 base pair by directional rotation and translation of the DNA duplex, and ADP and Pi release leads to translocation of the developing single strand.

338 citations