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Journal ArticleDOI

Fork sensing and strand switching control antagonistic activities of RecQ helicases

17 Jun 2013-Nature Communications (Nature Publishing Group)-Vol. 4, Iss: 1, pp 2024-2024

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.

AbstractRecQ 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.

Topics: Helicase (61%), Homologous recombination (51%), DNA replication (50%)

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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.

92 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.

72 citations


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.

57 citations


Journal ArticleDOI
TL;DR: A coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis is suggested, which further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase.
Abstract: Bacteriophage T7 gp4 serves as a model protein for replicative helicases that couples deoxythymidine triphosphate (dTTP) hydrolysis to directional movement and DNA strand separation. We employed single-molecule fluorescence resonance energy transfer methods to resolve steps during DNA unwinding by T7 helicase. We confirm that the unwinding rate of T7 helicase decreases with increasing base pair stability. For duplexes containing >35% guanine-cytosine (GC) base pairs, we observed stochastic pauses every 2–3 bp during unwinding. The dwells on each pause were distributed nonexponentially, consistent with two or three rounds of dTTP hydrolysis before each unwinding step. Moreover, we observed backward movements of the enzyme on GC-rich DNAs at low dTTP concentrations. Our data suggest a coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis, and they further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase.

51 citations


Additional excerpts

  • ...Ce and magnetic tweezers (Klaue et al., 2013; Qi et al., 2013)....

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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.

49 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)....

    [...]

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

    [...]


References
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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.
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Journal ArticleDOI
TL;DR: This Review discusses how these proteins might suppress genomic rearrangements, and therefore function as 'caretaker' tumour suppressors.
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402 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.
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314 citations